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ARTICLES

Induction of Programmed Cell Death in Kaposis


Sarcoma Cells by Preparations of Human Chorionic
Gonadotropin
Felipe Samaniego, Joseph L. Bryant, Ni Liu, Judith E. Karp, Anita L. Sabichi,
Alain Thierry, Yanto Lunardi-Iskandar, Robert C. Gallo

cells, which proliferate independently of cytokines (911) and


Background: Isolation of the first neoplastic acquired immu- are often clonal (12). We and others (11,12) have isolated tu-
nodeficiency syndrome-related Kaposis sarcoma (KS) cell morigenic cell lines (KS Y-1 and KS SLK) that are derived from
line (KS Y-1) has furthered understanding of the pathogen- nodular KS lesions, which contained rearrangement in chromo-
esis of KS. Studies with KS Y-1 cells have indicated that some 3 at region 3p14, the location of the transformation-related
inhibition of KS cell proliferation occurs in early pregnancy fragile histidine triad gene. Virtually all cases of KS tumors,
in mice and after treatment with certain commercial prepa- regardless of their epidemiologic type (acquired immunodefi-
rations of human chorionic gonadotropin (hCG, a pregnancy ciency syndrome [AIDS]-related, endemic, classical, or iatro-

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hormone purified from urine). The activity of the commer- genic), contain HHV8 DNA sequences suggesting that it is a
cial preparations has been attributed to an hCG-associated necessary cofactor (13), and when present with human immu-
factor(s) (HAF). While several clinical benefits of HAF are nodeficiency virus type 1 (HIV-1) infection, both contribute to a
clearly evident, the basis for its anti-KS properties remains progressive cancer.
unknown. We investigated the apoptosis-inducing effects of Our initial study (10) of tumors induced by the inoculation of
HAF and the expression of apoptosis-related proteins in KS KS Y-1 and KS SLK cells in nude mice revealed an unantici-
cells. Methods: KS Y-1 and KS SLK cells were treated with pated pronounced inhibition of tumor development during early
clinical-grade crude preparations of hCG, recombinant pregnancy. The anti-KS activity in mice was strikingly effective
hCG, or urine fractions exhibiting anti-KS activity and then during the first half of the gestation period when chorionic go-
examined for features of apoptosis. Levels of proteins asso- nadotropin-like activity in sera from mice (and humans) is maxi-
ciated with apoptosis were monitored by western blot analy- mum (the murine equivalent of human chorionic gonadotropin
sis, and cell DNA content was assessed by flow cytometry. [hCG] has not yet been identified). Some clinical-grade crude
Tumors induced in mice by inoculation of KS Y-1 cells were preparations of hCG initiated a regression of tumors induced by
treated with preparations of hCG, and the tumors were ex- KS Y-1 cells in nude mice and inhibited clonogenic KS Y-1 cell
amined for cell morphology and also for DNA fragmentation foci formation (10). These animal experiments spurred human
by use of the terminal deoxynucleotidyl transferase- trials to test local or systemic administration of hCG for KS.
Intralesional and systemic treatment with commercially avail-
mediated digoxigenin-deoxyuridine triphosphate nick-end-
able clinical-grade crude preparations of hCG induced signifi-
labeling (TUNEL) assay. Results: The HAF present in some
cant rates of regression of both cutaneous and of the more re-
preparations of hCG and in urine fractions has the ability to
sistant visceral KS lesions (14,15). Because commercial
induce apoptosis in KS cells in vitro and in vivo. HAF-
preparations of hCG from various sources contain variable anti-
triggered apoptosis was preceded by increased levels of the
KS activity yet equivalent endocrine activity and because highly
apoptosis-related proteins c-Myc and c-Rel and cell accumu-
purified hCG and recombinant hCG lack anti-KS activity, this
lation in G0/G1 phase of the cell cycle. KS Y-1 cells trans- collectively suggested to us that the activity of the commercial
fected with a c-Myc complementary DNA showed elevated preparations of hCG was due to a co-purified factor (or factors)
rates of apoptosis. Conclusion: The anti-KS activity of HAF that had the ability to induce regression of KS tumors. We have
appears to induce apoptosis. Such activity suggests a role for termed this anti-KS factor as hCG-associated factor(s) (HAF)
HAF in pregnancy-related regulation of cell death. [J Natl that is present in some, but not all, commercial clinical-grade
Cancer Inst 1999;91:13543]
Affiliations of authors: F. Samaniego, Institute of Human Virology, Medical
Kaposis sarcoma (KS) tumors are blood vessel-rich lesions Biotechnology Center and Greenebaum Cancer Center, University of Maryland,
characterized by proliferation of cytokine-dependent spindle Baltimore; J. L. Bryant, N. Liu, Y. Lunardi-Iskandar, R. C. Gallo, Institute of
cells, activated endothelial cells, immune cells (13), and the Human Virology, Medical Biotechnology Center, University of Maryland; J. E.
Karp, Greenebaum Cancer Center, University of Maryland; A. L. Sabichi, The
presence of human herpesvirus 8 (HHV8)-infected cells (4). University of Texas M. D. Anderson Cancer Center, Houston; A. Thierry, Trans-
These lesions express basic fibroblast growth factor (a potent gene, France.
angiogenic factor), platelet-derived growth factor, and vascular Correspondence to: Felipe Samaniego, M.D., Institute of Human Virology,
endothelial growth factor (5), as well as interleukin 6, hepato- Medical Biotechnology Center, University of Maryland, 725 W. Lombard St.,
cyte growth factor, and interleukin 8 that may collectively con- Rm. N454, Baltimore, MD 21201-1192 (e-mail: samanieg@umbi.umd.edu).
tribute to the abundantly vascularized appearance of these le- See Notes following References.
sions (68). Nodular or late-stage forms of KS harbor malignant Oxford University Press

Journal of the National Cancer Institute, Vol. 91, No. 2, January 20, 1999 ARTICLES 135
crude preparations of hCG and that which is present in urine KS4 cells are considered to represent the hyperplastic spindle cells of KS lesions.
during pregnancy. We have shown that anti-KS activity did not AIDS-KS4 cells as well as the two cell lines described above were maintained
reside in the hCG molecule or in one of its subunits (16). While in a gelatin (1.5%)-coated flask in RPMI-1640 medium containing the following:
15% fetal bovine serum (FBS), essential and nonessential amino acids, 1%
patients were undergoing treatment for KS with preparations of
Nutridoma (Boehringer Mannheim Corp., Indianapolis, IN), penicillin G (100
hCG, we also noted an improvement in their HIV-1 load, he- U/mL), and streptomycin (100 g/mL). These cells (KS Y-1, KS SLK, and
matopoietic parameters, and overall well-being. Furthermore, AIDS-KS4) lack HHV8 and HIV-1 DNA (17). Human umbilical vein endothe-
macaques with clinical simian immunodeficiency virus infection lial (H-UVE) cells and breast cancer cells (Bt-483) were included as controls;
treated with preparations of hCG showed reduced viral load, their propagation has been described by American Type Culture Collection
weight gain, and prevention of AIDS (16). The anti-HIV-1 and (Manassas, VA).
anti-KS activities appear to co-separate in partial purification of Partially purified HAF from urine of pregnant women. Urine from first-
HAF, suggesting that these activities may stem from the same trimester pregnancy (40 L) was filtered, concentrated, and then desalted on a
Sephadex G25 column that effectively reduced the volume to less than 500 mL
molecule(s) (16) but are yet to be proven conclusively. There-
(16). The protein-containing peak was then separated on a Superdex 200 column
fore, HAF may have the ability to reverse some of the effects of (26/60) (Pharmacia Biotech, Inc., Piscataway, NJ) in phosphate-buffered saline
AIDS, in addition to its anti-KS activity. While several clinical (PBS). Fractions were measured for total protein levels and for concentrations of
benefits are clearly evident with treatment with preparations of heterodimeric hCG and the hCG -core fragment (a cleaved form of hCG) by
hCG, the underlying basis for these improvements, including specific immunoassays. The hCG heterodimer elutes as a 70-kd molecule and the
anti-KS properties of HAF, remains unknown. To investigate the hCG -subunit core elutes as a 10- to 25-kd peak on gel filtration (16). hCG-
anti-KS activity of HAF, we treated KS cells in vitro and KS immunoreactivity peaked between fractions 46 and 49 of pregnancy urine con-
tumors in vivo with preparations of hCG and with partially pu- centrates. The first and second peaks of anti-KS activity elute with molecular
ranges of 1530 and 24 kd and their respective peak positions are remote from
rified fractions of HAF from the urine of pregnant women. We
the elution positions of hCG or hCG -free subunit.
describe apoptosis-related morphologic changes and protein ex- Treatment of cells with preparations of hCG and purified hCG (CR 127).

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pression. KS Y-1, KS SLK, and Bt-483 cells were seeded into T75 flasks at 20% con-
fluency in RPMI-1640 medium containing 15% FBS and grown for 1 day. The
METHODS cells were then synchronized by incubation for 24 hours in medium containing
1% FBS, pulsed for 24 hours with medium containing 10% FBS, and then
Treatment of tumors induced by KS Y-1 cells with preparations of hCG. incubated in medium containing 1% FBS, preparations of clinical-grade hCG, or
Tumors were first induced in beige nude mice (Harlem, Frederick, MD) by equivalent amounts of CR 127 (1001000 IU/mL, National Hormone and Pitu-
subcutaneous inoculation of 1 106 KS Y-1 cells into the dorsal lateral flanks, itary Program and Center for Population Research, National Institutes of Health,
which consistently produced 5 5 mm2 tumors within 2 weeks. Several com- Bethesda, MD), thyroid-stimulating hormone (TSH), a hCG-related heterodimer
mercial preparations of hCG (clinical-grade crude hCG at a dose of 500 IU [0.25 glycoprotein (Sigma Chemical Co., St. Louis, MO), or buffer containing the salts
IU/g protein]) were evaluated, but a batch of Wyeth Ayerst preparations of in preparations of hCG. The CR 127 was available in limited quantities and was
hCG (Wyeth Ayerst Laboratories, Philadelphia, PA) that induced apoptosis of used only in the preliminary experiments. To avoid cell death related to low FBS
KS Y-1 cells in culture was used in all studies except where shown otherwise. levels, H-UVE cells were cultured in RPMI-1640 medium with 10% FBS for 24
Preparations of hCG or an equivalent amount of hCG buffer (i.e., equivalent hours, then in medium containing 15% FBS for 24 hours, and finally with 10%
concentration of salts in commercial hCG preparations) were administered daily FBS and preparations of hCG or buffer control for the indicated intervals de-
by injection into KS lesions and adjacent tissues for 5 consecutive days. The scribed above. Cells were harvested for genomic DNA isolation (Stratagene, La
dose of 500 IU of commercial hCG was determined as an adequate dose required Jolla, CA) or for monitoring protein expression.
for inducing regression of the tumors, and a maximal tolerated dose was not Staining for actin and chromatin of cells treated with crude preparations
determined. Since the active principle with anti-KS activity is not totally char- of hCG, CR 127, or partially purified HAF fractions. One hundred thousand
acterized, any chance of variation in anti-KS activity among different prepara- KS Y-1 cells, KS SLK or AIDS-KS4 cells, or 104 H-UVE cells were seeded onto
tions was eliminated by using only one batch of hCG with reasonable activity. gelatinized glass chamber slides (200 mm2) and incubated for 24 hours with 500
Animal care was in accord with institutional guidelines. Bidirectional tumor IU/mL of preparations of hCG or buffer. The slides were fixed with formalin and
dimensions were noted before and after treatments, tumors were resected, and serially treated with Triton X-100 (0.01%) for 10 minutes at 25 C, with 0.4
hematoxylineosin-stained thin sections of tumors were examined for morphol- g/mL of fluorescein isothiocyanate (FITC)-labeled Phalloidin, which binds
ogy. To study the early effects of preparations of hCG-induced tumor regression, actin (Sigma Chemical Co.) for 30 minutes, washed with PBS, and then stained
tumors were treated intralesionally with preparations of hCG (500 IU) or buffer DNA with propidium iodide (0.5 g/mL) (Sigma Chemical Co.) for 15 minutes
for 2 days. The tumor sites and surrounding tissues were resected and fixed in (18). The stained slides were washed and mounted with Slow Fade (Molecular
formalin. The terminal deoxynucleotidyl transferase-mediated digoxigenin- Probes Inc., Eugene, OR) and examined (1000) through a dual emission filter
deoxyuridine triphosphate nick-end-labeling (TUNEL) assay was performed by XF 53 (Omega, Brattleboro, VA) by fluorescence microscopy. Also, KS Y-1
treatment of thin tissue slide sections with terminal deoxynucleotidyl transferase cells cultured in chamber slides were treated for 24 hours with clinical-grade
for in situ extension at DNA termini by incorporation of digoxigenin- crude preparations of hCG and equivalent amounts of CR127, recombinant hCG,
deoxyuridine triphosphate (Oncor Inc., Gaithersburg, MD). Cells with high lev- TSH, or with chromatographic fractions 48, 67, and 76 (the latter two contain
els of DNA termini support incorporation of digoxigenin-deoxyuridine triphos- anti-KS clonogenic activity) from human pregnancy urine concentrates (16), and
phate, which was detected by peroxidase-conjugated anti-digoxigenin antibody. fixed according to instructions described above, but without Triton X-100. The
Also, using the same tissue fixative method, thin tissue slides of control rat gut slides were then stained with propidium iodide and examined for chromatin and
were concurrently analyzed for apoptosis. Cells at the base and cells at the tip of cellular morphology under confocal microscopy.
rat gut villi tissue served as reference material for live and apoptotic cells, Protein expression in cells undergoing apoptosis after treatment with
respectively. preparations of hCG. KS Y-1, KS SLK, and H-UVE cells were synchronized
Cells. KS Y-1 cells were isolated and established as a neoplastic cell line from by pulsing with FBS and treated with clinical-grade crude preparations of hCG
a pleural effusion of an HIV-1-infected male with KS involving the lung (10), as described above. After 24 hours of incubation with preparations of hCG, the
and KS SLK cells were derived from an oral KS lesion of an HIV-1-noninfected cells were washed with PBS and lysed in buffer containing 1% Nonidet and the
kidney transplant recipient receiving immunosuppressive drugs (11). Both cell protease inhibitors leupeptin and aprotinin. The lysate was clarified by centrifu-
lines bear endothelial markers and induce angiogenic tumors when inoculated in gation (11 750g) at 4 C for 15 minutes. Clarified lysate (100 g protein) was
nude mice. AIDS-KS4 cells are early passage (passages 35) hyperplastic cells size separated by reducing 10% sodium dodecyl sulfatepolyacrylamide gel
directly derived from human KS lesions that express tissue specific markers, electrophoresis and transferred to nitrocellulose. Filters were blotted with anti-
proliferate in response to inflammatory cytokine stimulation and induce forma- bodies to c-Myc (9E10) (Santa Cruz Biotech, CA), retinoblastoma (RB) protein
tion of transient KS-like lesions when inoculated in nude mice (5,6,8). AIDS- (Santa Cruz Biotech), Bcl-2 (Dakopatts, Carpenteria, CA), and c-Rel (Santa Cruz

136 ARTICLES Journal of the National Cancer Institute, Vol. 91, No. 2, January 20, 1999
Biotech) or with respective isotype control antibody followed by incubation with
peroxidase-conjugated second antibody, and detection by chemiluminesence
analysis (Amersham Life Science Inc., Arlington Heights, IL). To determine
whether HAF-induced cellular proteins can by themselves induce cell death, Fig. 1. DNA isolated from tumors
c-Myc was expressed in cells using plasmid LTRhmyc. KS Y-1 cells (1 107) treated with preparations of human
were transfected with pLTRhmyc or vector alone (19) by electroporation (Gene chorionic gonadotropin (hCG) or
Pulser; Bio-Rad Laboratories, Hercules, CA) and monitored for c-Myc expres- with buffer. Tumors were induced in
sion by western blot analysis (using 9E10 antibody), and for cell death by beige nude mice by the inoculation of
morphology as described for preparations of hCG-treated cells. Cells were also Kaposis sarcoma (KS) Y-1 and KS
synchronized with FBS as described above and analyzed for apoptosis after SLK cells. Genomic DNA was iso-
incubation for 48 h with either 0100 ng/mL anti-Fas antibody or isotype control lated from tumors and mouse tissues
antibody (Upstate Biotechnology, Inc., Lake Placid, NY). 48 hours after intralesional treatment
Fluorescence flow cytometry. Preparations of hCG- or buffer-treated KS Y-1 with preparations of hCG or buffer.
cells were dislodged from monolayer cultures by gentle pipetting with cell Five micrograms of genomic DNA
dissociation buffer (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD). was size fractionated in a 1.2% aga-
To measure DNA content, cells were washed in PBS and fixed in 2% paraform- rose gel and stained with ethidium
aldehyde for 10 minutes at 4 C followed by ethanol/PBS (70%) for 2 hours at bromide.
20 C. The fixative was removed by centrifugation and the cells were washed
twice in PBS, incubated with ribonuclease A (10 g/mL) (Sigma Chemical Co.)
for 20 minutes at 37 C, stained with propidium iodide (5 g/mL) at 28 C, and
cells scanned by flow cytometry (see below). For detection of proteins, the
paraformaldehyde (2%) fixed and ethanol-treated cells were incubated with an-
tibodies targeting c-Myc (9E10) (Santa Cruz Biotech), RB (Santa Cruz Biotech),
Bcl-2 (Dakopatts), c-Rel (Santa Cruz Biotech), Fas (CH-11, Upstate Biotech- there was evidence that the regression of KS tumors induced by

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nology, Inc.), or their respective isotype control antibody for 30 minutes in PBS
the preparations of hCG was associated with oligonucleosomal
and then incubated with FITC-labeled second antibody (Boehringer Mannheim
Corp.). Baseline activity was assigned by staining cells with isotype control DNA fragmentation.
antibody. Analysis for DNA content and protein expression was accomplished To analyze the early mechanism leading to tumor regression,
on a FACScan flow cytometer using Lysis II software (Becton Dickinson, Ruth- formalin-fixed slides of tumor tissues were stained with hema-
erford, NJ). toxylineosin for morphologic analysis. Tumors treated with
Statistical analysis. Where appropriate, the means are shown with standard preparations of hCG showed hypocellular areas (Fig. 2, A, X)
deviation and representative experiments presented. The Students t test was
with a few densely staining cells, but no inflammatory cell in-
applied to estimate the statistical significance of difference (20). All P values
were two-sided. filtration. In contrast, surrounding normal mouse cells (Fig. 2, A,
Z) did not show depletion of cells or did not show cell mor-
RESULTS phology suggestive of an occurrence of apoptosis despite
equivalent exposure of tissues to preparations of hCG. To further
Oligonucleosomal DNA Cleavage and In Situ Apoptosis in assess the mechanism of tumor regression induced by prepara-
KS Tumors After Treatment With Preparations of hCG
tions of hCG, tumors were analyzed for the presence of DNA
To demonstrate the effects of clinical-grade crude hCG (HAF termini by using digoxigenin-deoxyuridine triphosphate nucleo-
in the preparations of hCG), KS tumors of equal size (5 5 mm) tide incorporation in TUNEL assay (Fig. 2, B, X) (9). Peroxidase
induced by the inoculation of KS Y-1 cells in beige nude mice activity (indicative of the presence of DNA termini) was mark-
were treated intralesionally with preparations of hCG (500 IU) edly evident within residual small cells in hypocellular areas of
or buffer daily for 5 days. The buffer-treated lesions in three tumors treated with preparations of hCG. Notably, there was no
mice showed continued tumor progression (>5 5 mm), extracellular staining, indicating that the DNA termini generated
whereas three mice treated with intralesional preparations of were retained in cells or intact cell fragments, which is consis-
hCG showed near-complete tumor regression (except that acel- tent with apoptosis and not necrosis, to account for depletion of
lular bands of tissue remained) confirming the anti-KS effect of cells. The aforementioned cell-associated high peroxidase activ-
preparations of hCG observed in previous studies (10). To ex- ity and morphologic changes were not observed in mouse tissues
amine the early effects of preparations of hCG on tumors, KS similarly treated with preparations of hCG (Fig. 2, B, Z) or in
Y-1 and KS SLK cell-induced tumors and surrounding mouse buffer-treated tumors (Fig. 2, C). Specifically, the treated tumors
tissues were treated with preparations of hCG (500 IU) or with contain areas showing on average (standard deviation [SD])
buffer for 2 days. On the third day the tumor sites and surround- 6.5% (1.3%), 7.3% (2.2%), 7.5% (1.9%), and 8.0% (1.4%)
ing tissues and other control tissues were resected and genomic positive staining cells per high-power field, whereas prepara-
DNA extracted. DNA isolated from buffer-treated tumors and tions of hCG-treated adjacent mouse tissues showed 1%
from endothelium-rich tissue (aorta) treated with preparations of (0.8%), 1% (0.8%), 0.2% (0.5%), and 0.5% (1%) positive
hCG (Fig. 1) showed high-molecular-weight DNA (>23 kilo- cells per high-power field, indicating significantly higher apop-
base) as determined on ethidium bromide-stained agarose gel. totic rates in treated tumors (P<.05). Buffer-treated tumors con-
The DNA of tumors treated with preparations of hCG showed tained 2.5% (1.3%) and 2% (1.4%) positive cells per high-
DNA in an oligonucleosomal fragmentation pattern typi- power field (Fig. 2, C). Control rat gut tissue sections
cal of programmed cell death (Fig. 1). Some of the preparations concurrently stained in TUNEL assay showed the presence and
of hCG-treated tumors showed minimal detectable fragmented absence of apoptosis indicated by peroxidase labeling in cells
DNA that was not easily visualized (Fig. 1, left lane; KS Y-1 residing in the tip and the base of villi, respectively (Fig. 2, D).
with 500 IU) while the TUNEL assay showed enhanced apop- Thus, treated tumors showed significantly higher rates of apop-
tosis (see below), indicating that detection of apoptosis by ge- tosis over control buffer-treated tumors. These observations ac-
nomic DNA examination was a less-sensitive technique. Thus, count for HAF-related KS lesion regression.

Journal of the National Cancer Institute, Vol. 91, No. 2, January 20, 1999 ARTICLES 137
Fig. 2. Tumors induced in nude mice by inocu-
lation of Kaposis sarcoma (KS) Y-1 cells un-
dergo apoptosis after treatment with preparations
of human chorionic gonadotropin (hCG). Repre-
sentative micrographs of sections of preparations
of hCG-treated and buffer-treated KS Y-1 tumor
in nude mice are shown. A) Shows hypocellular
area (X) with pyknotic nuclei. These morpho-
logic changes were not observed in unaffected
mouse tissue (Z). B) The similarly treated tis-
sues analyzed for the presence of DNA termini
by terminal deoxynucleotidyl transferase-
mediated digoxigenin deoxyuridine triphosphate
nick-end-labeling (TUNEL) assay show some
tumor cells (X) staining strongly positive with
the smallest cell bodies containing more intense
activity, whereas preparations of hCG-treated
mouse tissues (Z) did not show activity. C)
Buffer-treated tumor and mouse tissues showed
much reduced rates of apoptosis by the TUNEL
assay. D) For positive and negative results, rat
gut in TUNEL assay shows positive and nega-
tive cells at the tip and at the base of villi, re-
spectively. The brown immunoperoxidase reac-

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tion product indicates positive detection of
significant levels of DNA termini.

DNA Fragmentation in KS Y-1 and KS SLK Cells Treated nuclear size ratio (Fig. 4, AD). Buffer-treated cells showed
With Preparations of hCG sharply defined nucleoli and well-defined filamentous actin of
the cytoskeleton that was distributed widely throughout the cy-
To determine whether the anti-KS activity of preparations of
toplasm. In contrast, KS Y-1, KS SLK, and AIDS-KS4 cells
hCG is a direct effect, tumor and control cells in culture were
treated with preparations of hCG (Fig. 4, EG) exhibited overall
treated with preparations of hCG (400 IU/mL) for 48 hours.
smaller cell size and contained small nuclei with focal areas of
Genomic DNA was isolated from cells and size-fractionated by
intense homogeneous nuclear staining. Similarly treated H-UVE
electrophoresis. The DNA from KS Y-1, KS SLK, and AIDS-
KS4 cells, after treatment with preparations of hCG, was frag- cells did not show the morphologic changes found in KS Y-1
mented in an oligonucleosomal pattern, whereas DNA from cells treated with preparations of hCG (Fig. 4, H) as observed by
similarly treated control H-UVE (21) and Bt 483 cells, as well as others (21). This acquired morphology is found in the earliest
buffer-treated KS cells, was of high molecular weight (Fig. 3). recognizable phases of apoptosis (18). In addition, cells treated
Treatment of KS Y-1 and KS SLK cells with highly purified with preparations of hCG displayed a low cytoplasmic-to-
hCG (CR 127) did not induce cell death (see below) suggesting nuclear size ratio and a reduced content of filamentous actin that
selective HAF activity in clinical-grade crude preparations of was poorly defined and preferentially retracted to the perinuclear
hCG was not due to hCG. area as observed during early apoptosis (18). Moreover, exami-
nation by confocal microscopy, revealed a twofold to fourfold
Morphologic Changes Induced by Preparations of hCG reduction in the cross-sectional nuclear area and in the overall
To characterize (with enhanced resolution) the early steps cell area of treated KS Y-1 cells compared with buffer-treated KS
accompanying apoptosis, cells were monitored by epifluores- Y-1 cells. Thus, HAF in clinical-grade crude preparations of hCG
cence microscopy 24 hours after treatment with preparations of induces prototypical early cell morphology changes of apoptosis.
hCG. Fig. 4 shows representative morphology of the buffer- To begin to identify HAF that is present in human pregnancy
treated cells, which contain a relatively high, cytoplasmic-to- urine, KS Y-1 cells were selected for testing the limited avail-
able HAF material isolated in chromatography-purified fractions
from urine. The KS Y-1 cells were treated with HAF fractions
for 48 hours and cells labeled with propidium iodide. Commer-
Fig. 3. Preparations of human chorionic
gonadotropin (hCG) induce oligonucleo-
cial preparations of hCG (Fig. 5, B) and urine HAF fractions 67
somal DNA fragmentation of Kaposis sar- (Fig. 5, E) and 76 (Fig. 5, F) induced reduction in cell size and
coma (KS) cells in culture. KS Y-1 and KS diffuse staining and clumping of chromatin, which is consistent
SLK cells, acquired immunodeficiency with apoptosis. Insufficient material in HAF fractions 67 and 76
syndrome (AIDS)-KS and control primary prevented further evaluation of these fractions. In contrast,
cells, human umbilical vein endothelial (H- buffer treatment (Fig. 5, A), CR 127 (Fig. 5, C), and urine
UVE) and breast cancer (Bt-483) cells
fraction 48 (Fig. 5, D) did not acquire morphology of apoptotic
were treated for 48 h with preparations of
hCG 400 IU/mL (+) or buffer (). Geno- cells even with a much higher concentration (188 g/mL), sug-
mic DNA was isolated and electrophoreti- gesting specific anti-KS activity in fractions 67 and 76. In ad-
cally separated in a 1.2 % agarose gel and ditional experiments, treatment of KS SLK and KS Y-1 cells
stained with ethidium bromide. with CR 127, recombinant hCG, or TSH did not induce changes
in cell number over 5 days, indicating lack of anti-KS activity.

138 ARTICLES Journal of the National Cancer Institute, Vol. 91, No. 2, January 20, 1999
Fig. 4. Preparations of human chorionic gonadotropin (hCG) induce morpho- with preparations of hCG (H) contain a high cytoplasm to nuclear ratio and
logic alterations consistent with apoptosis. Cells were seeded in chamber slides well-defined filamentous actin distributed widely throughout cytoplasm. In con-
and incubated with buffer or preparations of hCG for 24 hours. The cells were trast, Kaposis sarcoma (KS) Y-1, KS SLK, and acquired immunodeficiency

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fixed and serially stained for actin and chromatin with fluorescein isothiocya- syndrome (AIDS)-KS4 cells treated with preparations of hCG (E-G) exhibited
nate-labeled Phalloidin and propidium iodide, respectively, and observed under contraction of cytoplasm, accumulation of poorly defined filaments in the peri-
fluorescence microscopy as outlined in the Methods section. The buffer- nuclear area, and a lower cytoplasm to nucleus ratio, all changes characteristic
treated cells (A-D) and human umbilical vein endothelial (H-UVE) cells treated of early apoptotic changes (18).

Fig. 5. Pregnancy urine-derived human chorionic gonadotropin


(hCG)-associated factor (HAF), preparations of hCG (clinical-
grade crude), but not highly purified hCG, induce apoptosis of
Kaposis sarcoma (KS) Y-1 cells as visualized through confocal
microscopy. A) KS Y-1 cells were treated with buffer, B) with
clinical-grade preparations of hCG (Wyeth Ayerst Laboratories,
Philadelphia, PA), C) CR 127, D) or partially purified HAF from
urine fractions 48, E) fraction 67, or F) fraction 76, and stained with
propidium iodide. Panels show normal heterogeneous DNA stain-
ing of large nuclei in cells treated with buffer, CR 127 and urine
fraction 48. In contrast, cells treated with preparations of hCG,
partially purified HAF from urine fractions 67 or 76 showed ho-
mogeneous DNA staining in a clump pattern in small nuclei. After
incubation of cells for 24 hours with the different treatments, the
cells were fixed and stained with propidium iodide and visualized
under confocal microscopy as outlined in the Methods section.

Fractions 67 and 76 also abrogated formation of KS Y-1 cell foci ipate that preparations of hCG-stimulated proliferation-
in clonogenic assays, as shown previously (16). Fractions 67 and associated pathways in KS cells preferentially operate in these
76 do not contain hCG heterodimer, hCG subunits, or -core of cells to accomplish programmed cell death (2126). After treat-
hCG. Thus, partial purification of HAF from urine of pregnant ment with clinical-grade crude preparations of hCG (400 IU/
women contains apoptosis-inducing factor(s) that are not attrib- mL) for 24 hours, extracts of KS Y-1 and KS SLK cells were
uted to hCG, its subunits, or -core of hCG. examined for proliferation-associated protein expression by
western blot analysis. While buffer-treated cells and cells with
Protein Expression in Apoptosis Induced by Preparations
no additional treatment contained undetectable levels of c-Myc,
of hCG
both KS Y-1 and KS SLK cells when treated with preparations
Because preparations of hCG are known to stimulate the of hCG showed markedly enhanced levels of c-Myc (Fig. 6, A).
growth of some cells and induce apoptosis in others, we antic- In parallel studies, treatment of KS Y-1 cells with preparations

Journal of the National Cancer Institute, Vol. 91, No. 2, January 20, 1999 ARTICLES 139
examined for cell death by following the same protocols for
preparations of hCG treatment of cells. Whereas the three
LTRhmyc-transfected pools contained an average (SD) of 23
(13.1) cells per high-power field with morphology consistent
with apoptosis, the vector-alone transfected cells and mock-
transfected cells contained 5.2 (6.4) and 4 (8.1) apoptotic cells
per high-power field, respectively. Thus, induced c-Myc expres-
sion stimulates apoptosis, suggesting that c-Myc may participate
in programmed cell death in KS cells after treatment with prepa-
rations of hCG.
KS Y-1 and KS SLK cells (Fig. 7, A and B, respectively)
were found to express modest levels of Fas, as shown by the
shift in black peak from white peak (isotype antibody control)
with anti-fas antibody. We tested whether these cells were sus-
ceptible to Fas-mediated cell killing by treating cells with anti-
Fas antibody (0100 L of CH-11) that induces apoptosis but
did not observe enhanced apoptosis (<2% apoptotic cells in both
antibody-treated cells and isotype control antibody-treated cells).
Given these negative results, it is likely that HAF-mediated apop-
tosis involves mechanisms independent of Fas receptor stimulation.
In a second strategy to analyze protein expression in HAF-

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induced apoptosis, cells were monitored for DNA and proto-
Fig. 6. Preparations of human chorionic gonadotropin (hCG) induce expression oncogene content by flow cytometry analysis. KS Y-1 cells
of c-Myc and c-Rel and accumulation of the hypophosphorylated form of the treated with preparations of hCG and stained with propidium
retinoblastoma (RB) protein. A) Treatment of cells with preparations of hCG iodide contained a subset of cells with hypodiploid levels of
(400 IU/mL) induced elevated c-Myc protein levels (arrow) in the Kaposis DNA content (Fig. 7, D) that was absent in buffer-treated cells
sarcoma (KS) Y-1 and KS SLK cell lines but not in endothelial cells (data not
(Fig. 7, C). The cells with hypodiploid DNA content were of
shown). B) Preparations of hCG treatment (400 IU/mL) also induced KS Y-1
cells to express c-Rel protein. Similar analyses show that preparations of hCG relatively smaller size as shown by the epifluorescence studies
induced a reduction of the levels of Bcl-2 protein and no changes in the p53 described above and findings are consistent with an apoptotic
protein levels (data not shown). Preparations of hCG treatment also induced mechanism. The cells treated with preparations of hCG also
hypophosphorylation of the RB protein as shown by the preferential loss of the preferentially accumulated in G0/G1 phase (Fig. 7, D). These
higher molecular weight band (RB). KS Y-1 and KS SLK cells were treated with observations are in agreement with accumulation of hypophos-
preparations of hCG (200 or 400 IU/mL) or buffer for 24 hours and lysates of phorylated RB protein (Fig. 6, B). Antibody staining of cells
cells were analyzed by western blots using the indicated antibodies as described
in the Methods section.
treated with preparations of hCG exhibited a higher number of
cells expressing c-Myc (37%78%), c-Rel (27%48%), and un-
changed levels of RB protein (Table 1). In addition, treated cells
contained fewer cells (by 50%) expressing Bcl-2. These results
of hCG (400 IU/mL) induced c-Rel protein expression, indicat-
indicate coordinated protein expression as well as cell cycle
ing a coordinated expression of at least two proto-oncogenes
modulation in HAF-induced programmed cell death.
known to participate in the same signaling pathway (23) (Fig. 6,
B). Additionally, KS Y-1 cell expression of RB protein did not DISCUSSION
show changes in the overall levels of RB after treatment with
preparations of hCG (data not shown). However, there was a Our initial observation linking anti-KS activity with factors
preferential reduction in the hyperphosphorylated form (higher associated with early pregnancy in mice (10) has led to these
molecular weight of the two bands) of RB in KS Y-1 cells (Fig. studies that attribute novel apoptotic HAF activity present in
6, B) after treatment with preparations of hCG. The diminished some clinical-grade crude preparations of hCG (which is com-
hyperphosphorylated levels of RB is consistent with accumula- mercially prepared from urine of pregnant women) and in urine
tion of cells in G0/G1 phase and this type of cell cycle regulation concentrates from pregnant women prepared in our laboratory.
is characteristic of certain apoptotic pathways (27). Moreover, Here, we show HAF induces KS cell programmed cell death in
western blot analysis showed that treatment with preparations of cell culture and in vivo as demonstrated by oligonucleosomal
hCG lowered Bcl-2 levels (by 50%), whereas the levels of p53 DNA fragmentation and the selective accumulation of abundant
remained unchanged (data not shown), as commonly noted in DNA termini in individual tumor cells in culture and in a KS
programmed cell death induced by the nongenotoxic type of tumor in an animal model. Cells treated with preparations of
stimuli (22). Thus, these data indicate HAF ultimately induces hCG undergo typical cellular morphologic changes that are fea-
apoptosis following expression of proliferation-associated pro- tures of cells that undergo apoptosis. This includes nuclear con-
teins as observed with other mitogen-triggered apoptosis. densation and global cell shrinkage with dissolution of the pe-
To better determine c-Myc expression in the apoptotic pro- ripheral actin cytoskeleton (18). Apoptosis follows up-regulated
cess induced by preparations of hCG, we examined whether c-Myc and c-Rel protein levels and a simultaneous reduction of
c-Myc expression in itself was sufficient to stimulate pro- Bcl-2 levels that were observed both by western blot analysis
grammed cell death. To test this, KS Y-1 cells were electropor- and flow cytometry analysis. Aside from our initial studies de-
ated with pLTRhmyc and cells evaluated for c-Myc expression scribing the anti-KS effect of clinical-grade crude preparations
(at 24 hours) by western blot analysis. These pools of cells were of hCG, others (21) have noted KS cell apoptosis with nonclini-

140 ARTICLES Journal of the National Cancer Institute, Vol. 91, No. 2, January 20, 1999
Fig. 7. Kaposis sarcoma (KS) Y-1 and KS SLK
cells express Fas and preparations of human chori-
onic gonadotropin (hCG) induce apoptosis by flow
cytometry analysis. A) KS Y-1 cells and B) KS SLK
cells express modest levels of Fas by flow cytometry
analysis. The open peak indicates activity with iso-
type control antibody whereas the black (filled peak)
indicates activity with anti-Fas antibody. Prepara-
tions of hCG treatment reduce the DNA content of
KS Y-1 cells (C and D). Whereas buffer-treated
cells (C) contain more than 95% of cells with at least
2N DNA complement, the preparations of hCG-
treated cells (D) exhibit a substantial higher number
of cells with lower DNA content. Cells treated with
preparations of hCG also preferentially accumulate
in G0/G1. For Fas expression, the cells were fixed
and stained with anti-Fas antibody (CH-11) and sub-
sequently with fluorescein isothiocyanate-labeled
antibody as described in the Methods section.
For DNA content analysis, cells were treated with
buffer or preparations of hCG for 48 hours and were
fixed and incubated with propidium iodide as de-
scribed in the Methods section. The cells were
then monitored for DNA content by flow cytometry

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analysis. For panels C and D, the vertical axis indi-
cates the relative cell number and the horizontal axis
(left to right) is the relative DNA content of prop-
idium stained cells (low to high). Stained cells were-
analyzed by flow cytometry for expression of other
proteins as shown in Table 1.

Table 1. Preparations of human chorionic gonadotropin (hCG) modulates HAF-induced apoptosis (28,30). Even though c-Myc-related ap-
proto-oncogene levels in Kaposis sarcoma Y-1 cells undergoing apoptosis* optosis has extensively been described in cell culture, a study
Cells expressing protein, %
(25) implicates c-Myc in apoptosis as a mechanism in cancer
regression in vivo.
Protein Buffer Preparations of hCG The identity of HAF is unknown. Fractions of preparations of
c-Myc 37 78 hCG containing HAF activity are distinct from hCG, its sub-
c-Rel 27 48 units, or nick forms and is characterized as a protein by dena-
Bcl-2 46 22 turing and protein digest experiments (16). Others (31) have
Retinoblastoma protein 86 82
found ribonuclease isolated from preparations of hCG to contain
*Proto-oncogene expression in KS Y-1 cells treated with preparations of hCG
anti-KS activity; however, the purified fractions with HAF pre-
were examined by flow cytometry. KS Y-1 cells were treated with preparations pared in our laboratory lack ribonuclease. Fractions containing
of hCG (400 IU/mL) or buffer for 24 hours. The cells were fixed and stained HAF-induced apoptosis of KS cells were not blocked by antiri-
with antibodies for targeting proteins indicated and examined by flow cytometry bonuclease antibodies. Interestingly, hCG is susceptible to pro-
analysis as described in the Methods section. The results are from represen- teolytic cleavage in vivo and can potentially produce peptides
tative studies and indicate the percentage of positive staining cells over signal that activate receptors. The subunit of hCG can be
generated with control isotype antibodies.
potentially cleaved to form hCG AA 3757. Synthetic forms of
this peptide form amphipathic helical structures that can
cal-grade preparations of hCG, as well as reduced cell prolifera- mimic the hCG heterodimer in activating its cognate receptor,
tion (28). albeit at several-fold higher concentrations (32).
The newly described apoptotic effect attributed to HAF re- Since HAF activity is found in urine in early pregnancy (16),
sembles the apoptosis pathway best followed by a number of its presence may be linked to the substantially lower rates of
growth factors related to programmed cell death. In various cell AIDS-KS in women versus men (33). HIV-1-infected women
systems, extracellular ligands and hormones that stimulate cell have lower rates of HHV8 infection and KS compared with
growth pathways can also, under the restricted conditions, in- HIV-1-infected men. Even though nonpregnant women already
duce apoptosis. Because preparations of hCG can induce prolif- exhibit lower rates of KS, one would predict that pregnancy via
eration and/or induce c-Myc in some normal and neoplastic cells HAF would offer enhanced protection against development of
(26,29), it was anticipated to precipitate apoptosis, perhaps by KS or HHV8 infection. However, a single retrospective study by
diverting an activated cell proliferation pathway to a cell death Rabkin et al. (34) in a small number of HIV-1-infected African
end point (2325). Here, HAF-induced c-Myc and c-Rel expres- women found that pregnancy was not associated with lower KS
sion, as well as induced c-Myc expression in transfectants, was rates. For other types of cancers, such as mammary cancer in
followed by KS cell apoptosis, suggesting participation of these humans and rats, the pregnancy state does confer subsequent
transcription factors in HAF-induced cell death. Preparations of lower rates of tumor occurrence. During pregnancy, breast tissue
hCG-stimulated c-Myc and c-Rel concur with results from other undergoes differentiation that renders breast tissue cells resistant
laboratories showing regulation of AP-1 complex formation in to transformation (35). In contrast, the HAF effect we observed

Journal of the National Cancer Institute, Vol. 91, No. 2, January 20, 1999 ARTICLES 141
with KS cells and preparations of hCG is primarily an apoptotic Kaposis sarcoma in patients with and without HIV infection. N Engl J Med
mechanism. Although stimulated cell differentiation can subse- 1995;332:11815.
(14) Gill PS, Lunardi-Iskandar Y, Louie S, Tulpule A, Zheng T, Espina BM, et
quently include apoptosis in some cells (36) and a transient
al. The effects of preparations of human chorionic gonadotropin on
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immediate and primarily apoptotic effect. Indeed, apoptosis (15) Hermans P, Clumeck K, Picard O, Van Vooren JP, Duriez P, Zucman D,
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Journal of the National Cancer Institute, Vol. 91, No. 2, January 20, 1999 ARTICLES 143