Affiliations of authors: F. Samaniego, Institute of Human Virology, Medical Biotechnology Center and Greenebaum Cancer Center, University of Maryland, Baltimore; J. L. Bryant, N. Liu, Y. Lunardi-Iskandar, R. C. Gallo, Institute of Human Virology, Medical Biotechnology Center, University of Maryland; J. E. Karp, Greenebaum Cancer Center, University of Maryland; A. L. Sabichi, The University of Texas M. D. Anderson Cancer Center, Houston; A. Thierry, Transgene, France.
Correspondence to: Felipe Samaniego, M.D., Institute of Human Virology, Medical Biotechnology Center, University of Maryland, 725 W. Lombard St., Rm. N454, Baltimore, MD 21201-1192 (e-mail: samanieg{at}umbi.umd.edu).
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ABSTRACT |
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INTRODUCTION |
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Our initial study (10) of tumors induced by the inoculation of KS Y-1 and KS SLK cells in nude mice revealed an unanticipated pronounced inhibition of tumor development during early pregnancy. The anti-KS activity in mice was strikingly effective during the first half of the gestation period when chorionic gonadotropin-like activity in sera from mice (and humans) is maximum (the murine equivalent of human chorionic gonadotropin [hCG] has not yet been identified). Some clinical-grade crude preparations of hCG initiated a regression of tumors induced by KS Y-1 cells in nude mice and inhibited clonogenic KS Y-1 cell foci formation (10). These animal experiments spurred human trials to test local or systemic administration of hCG for KS. Intralesional and systemic treatment with commercially available clinical-grade crude preparations of hCG induced significant rates of regression of both cutaneous and of the more resistant visceral KS lesions (14,15). Because commercial preparations of hCG from various sources contain variable anti-KS activity yet equivalent endocrine activity and because highly purified hCG and recombinant hCG lack anti-KS activity, this collectively suggested to us that the activity of the commercial preparations of hCG was due to a co-purified factor (or factors) that had the ability to induce regression of KS tumors. We have termed this anti-KS factor as hCG-associated factor(s) (HAF) that is present in some, but not all, commercial clinical-grade crude preparations of hCG and that which is present in urine during pregnancy. We have shown that anti-KS activity did not reside in the hCG molecule or in one of its subunits (16). While patients were undergoing treatment for KS with preparations of hCG, we also noted an improvement in their HIV-1 load, hematopoietic parameters, and overall well-being. Furthermore, macaques with clinical simian immunodeficiency virus infection treated with preparations of hCG showed reduced viral load, weight gain, and prevention of AIDS (16). The anti-HIV-1 and anti-KS activities appear to co-separate in partial purification of HAF, suggesting that these activities may stem from the same molecule(s) (16) but are yet to be proven conclusively. Therefore, HAF may have the ability to reverse some of the effects of AIDS, in addition to its anti-KS activity. While several clinical benefits are clearly evident with treatment with preparations of hCG, the underlying basis for these improvements, including anti-KS properties of HAF, remains unknown. To investigate the anti-KS activity of HAF, we treated KS cells in vitro and KS tumors in vivo with preparations of hCG and with partially purified fractions of HAF from the urine of pregnant women. We describe apoptosis-related morphologic changes and protein expression.
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METHODS |
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Cells. KS Y-1 cells were isolated and established as a neoplastic cell line from a pleural effusion of an HIV-1-infected male with KS involving the lung (10), and KS SLK cells were derived from an oral KS lesion of an HIV-1-noninfected kidney transplant recipient receiving immunosuppressive drugs (11). Both cell lines bear endothelial markers and induce angiogenic tumors when inoculated in nude mice. AIDS-KS4 cells are early passage (passages 3-5) hyperplastic cells directly derived from human KS lesions that express tissue specific markers, proliferate in response to inflammatory cytokine stimulation and induce formation of transient KS-like lesions when inoculated in nude mice (5,6,8). AIDS-KS4 cells are considered to represent the hyperplastic spindle cells of KS lesions. AIDS-KS4 cells as well as the two cell lines described above were maintained 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% Nutridoma (Boehringer Mannheim Corp., Indianapolis, IN), penicillin G (100 U/mL), and streptomycin (100 µg/mL). These cells (KS Y-1, KS SLK, and AIDS-KS4) lack HHV8 and HIV-1 DNA (17). Human umbilical vein endothelial (H-UVE) cells and breast cancer cells (Bt-483) were included as controls; their propagation has been described by American Type Culture Collection (Manassas, VA).
Partially purified HAF from urine of pregnant women. Urine from first-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 (16). The protein-containing peak was then separated on a Superdex 200 column (26/60) (Pharmacia Biotech, Inc., Piscataway, NJ) in phosphate-buffered saline (PBS). Fractions were measured for total protein levels and for concentrations of heterodimeric hCG and the hCG ß-core fragment (a cleaved form of hCG) by specific immunoassays. The hCG heterodimer elutes as a 70-kd molecule and the hCG ß-subunit core elutes as a 10- to 25-kd peak on gel filtration (16). hCG-immunoreactivity peaked between fractions 46 and 49 of pregnancy urine concentrates. The first and second peaks of anti-KS activity elute with molecular ranges of 15-30 and 2-4 kd and their respective peak positions are remote from the elution positions of hCG or hCG ß-free subunit.
Treatment of cells with preparations of hCG and purified hCG (CR 127). KS Y-1, KS SLK, and Bt-483 cells were seeded into T75 flasks at 20% confluency in RPMI-1640 medium containing 15% FBS and grown for 1 day. The 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 incubated in medium containing 1% FBS, preparations of clinical-grade hCG, or equivalent amounts of CR 127 (100-1000 IU/mL, National Hormone and Pituitary Program and Center for Population Research, National Institutes of Health, Bethesda, MD), thyroid-stimulating hormone (TSH), a hCG-related heterodimer glycoprotein (Sigma Chemical Co., St. Louis, MO), or buffer containing the salts in preparations of hCG. The CR 127 was available in limited quantities and was used only in the preliminary experiments. To avoid cell death related to low FBS levels, H-UVE cells were cultured in RPMI-1640 medium with 10% FBS for 24 hours, then in medium containing 15% FBS for 24 hours, and finally with 10% FBS and preparations of hCG or buffer control for the indicated intervals described above. Cells were harvested for genomic DNA isolation (Stratagene, La Jolla, CA) or for monitoring protein expression.
Staining for actin and chromatin of cells treated with crude preparations of hCG, CR 127, or partially purified HAF fractions. One hundred thousand KS Y-1 cells, KS SLK or AIDS-KS4 cells, or 104 H-UVE cells were seeded onto gelatinized glass chamber slides (200 mm2) and incubated for 24 hours with 500 IU/mL of preparations of hCG or buffer. The slides were fixed with formalin and serially treated with Triton X-100 (0.01%) for 10 minutes at 25 °C, with 0.4 µg/mL of fluorescein isothiocyanate (FITC)-labeled Phalloidin, which binds actin (Sigma Chemical Co.) for 30 minutes, washed with PBS, and then stained DNA with propidium iodide (0.5 µg/mL) (Sigma Chemical Co.) for 15 minutes (18). The stained slides were washed and mounted with Slow Fade (Molecular Probes Inc., Eugene, OR) and examined (1000x) through a dual emission filter XF 53 (Omega, Brattleboro, VA) by fluorescence microscopy. Also, KS Y-1 cells cultured in chamber slides were treated for 24 hours with clinical-grade crude preparations of hCG and equivalent amounts of CR127, recombinant hCG, TSH, or with chromatographic fractions 48, 67, and 76 (the latter two contain anti-KS clonogenic activity) from human pregnancy urine concentrates (16), and fixed according to instructions described above, but without Triton X-100. The slides were then stained with propidium iodide and examined for chromatin and cellular morphology under confocal microscopy.
Protein expression in cells undergoing apoptosis after treatment with preparations of hCG. KS Y-1, KS SLK, and H-UVE cells were synchronized by pulsing with FBS and treated with clinical-grade crude preparations of hCG as described above. After 24 hours of incubation with preparations of hCG, the cells were washed with PBS and lysed in buffer containing 1% Nonidet and the protease inhibitors leupeptin and aprotinin. The lysate was clarified by centrifugation (11 750g) at 4 °C for 15 minutes. Clarified lysate (100 µg protein) was size separated by reducing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Filters were blotted with antibodies to c-Myc (9E10) (Santa Cruz Biotech, CA), retinoblastoma (RB) protein (Santa Cruz Biotech), Bcl-2 (Dakopatts, Carpenteria, CA), and c-Rel (Santa Cruz 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, c-Myc was expressed in cells using plasmid LTRhmyc. KS Y-1 cells (1 x 107) were transfected with pLTRhmyc or vector alone (19) by electroporation (Gene Pulser; Bio-Rad Laboratories, Hercules, CA) and monitored for c-Myc expression by western blot analysis (using 9E10 antibody), and for cell death by morphology as described for preparations of hCG-treated cells. Cells were also synchronized with FBS as described above and analyzed for apoptosis after incubation for 48 h with either 0-100 ng/mL anti-Fas antibody or isotype control antibody (Upstate Biotechnology, Inc., Lake Placid, NY).
Fluorescence flow cytometry. Preparations of hCG- or buffer-treated KS Y-1 cells were dislodged from monolayer cultures by gentle pipetting with cell dissociation buffer (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD). To measure DNA content, cells were washed in PBS and fixed in 2% paraformaldehyde for 10 minutes at 4 °C followed by ethanol/PBS (70%) for 2 hours at -20 °C. The fixative was removed by centrifugation and the cells were washed twice in PBS, incubated with ribonuclease A (10 µg/mL) (Sigma Chemical Co.) for 20 minutes at 37 °C, stained with propidium iodide (5 µg/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 antibodies targeting c-Myc (9E10) (Santa Cruz Biotech), RB (Santa Cruz Biotech), Bcl-2 (Dakopatts), c-Rel (Santa Cruz Biotech), Fas (CH-11, Upstate Biotechnology, Inc.), or their respective isotype control antibody for 30 minutes in PBS and then incubated with FITC-labeled second antibody (Boehringer Mannheim Corp.). Baseline activity was assigned by staining cells with isotype control antibody. Analysis for DNA content and protein expression was accomplished on a FACScan flow cytometer using Lysis II software (Becton Dickinson, Rutherford, NJ).
Statistical analysis. Where appropriate, the means are shown with standard deviation and representative experiments presented. The Student's t test was applied to estimate the statistical significance of difference (20). All P values were two-sided.
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RESULTS |
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To demonstrate the effects of clinical-grade crude hCG (HAF in the
preparations of hCG), KS tumors of equal size (5 x 5 mm) induced by
the inoculation of KS Y-1 cells in beige nude mice were treated
intralesionally with preparations of hCG (500 IU) or buffer daily for 5
days. The buffer-treated lesions in three mice showed continued tumor
progression (>5 x 5 mm), whereas three mice treated with
intralesional preparations of hCG showed near-complete tumor regression
(except that acellular bands of tissue remained) confirming the anti-KS
effect of preparations of hCG observed in previous studies
(10). To examine the early effects of preparations of hCG on
tumors, KS Y-1 and KS SLK cell-induced tumors and surrounding mouse
tissues were treated with preparations of hCG (500 IU) or with buffer
for 2 days. On the third day the tumor sites and surrounding tissues
and other control tissues were resected and genomic DNA extracted. DNA
isolated from buffer-treated tumors and from endothelium-rich tissue
(aorta) treated with preparations of hCG (Fig. 1)
showed high-molecular-weight DNA (>23 kilobase) as determined on
ethidium bromide-stained agarose gel. The DNA of tumors treated with
preparations of hCG showed DNA in an oligonucleosomal fragmentation
pattern typical of programmed cell death (Fig. 1).
Some of the
preparations of hCG-treated tumors showed minimal detectable fragmented
DNA that was not easily visualized (Fig. 1,
left lane; KS Y-1 with 500
IU) while the TUNEL assay showed enhanced apoptosis (see
below), indicating that detection of apoptosis by genomic DNA
examination was a less-sensitive technique. Thus, there was evidence
that the regression of KS tumors induced by the preparations of hCG was
associated with oligonucleosomal DNA fragmentation.
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To determine whether the anti-KS activity of preparations of hCG is
a direct effect, tumor and control cells in culture were treated with
preparations of hCG (400 IU/mL) for 48 hours. Genomic DNA was isolated
from cells and size-fractionated by electrophoresis. The DNA from KS
Y-1, KS SLK, and AIDS-KS4 cells, after treatment with preparations of
hCG, was fragmented in an oligonucleosomal pattern, whereas DNA from
similarly treated control H-UVE (21) and Bt 483 cells, as well
as buffer-treated KS cells, was of high molecular weight (Fig.
3). Treatment of KS Y-1 and KS SLK cells with highly
purified hCG (CR 127) did not induce cell death (see below)
suggesting selective HAF activity in clinical-grade crude preparations
of hCG was not due to hCG.
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To characterize (with enhanced resolution) the early steps
accompanying apoptosis, cells were monitored by epifluorescence
microscopy 24 hours after treatment with preparations of hCG. Fig.
4 shows representative morphology of the
buffer-treated cells, which contain a relatively high,
cytoplasmic-to-nuclear size ratio (Fig. 4, A-D).
Buffer-treated cells
showed sharply defined nucleoli and well-defined filamentous actin of
the cytoskeleton that was distributed widely throughout the cytoplasm.
In contrast, KS Y-1, KS SLK, and AIDS-KS4 cells treated with
preparations of hCG (Fig. 4, E-G)
exhibited overall smaller cell size
and contained small nuclei with focal areas of intense homogeneous
nuclear staining. Similarly treated H-UVE cells did not show the
morphologic changes found in KS Y-1 cells treated with preparations of
hCG (Fig. 4, H)
as observed by others (21). This
acquired morphology is found in the earliest recognizable phases of apoptosis
(18). In addition, cells treated with preparations of hCG
displayed a low cytoplasmic-to-nuclear size ratio and a reduced content
of filamentous actin that was poorly defined and preferentially
retracted to the perinuclear area as observed during early apoptosis
(18). Moreover, examination by confocal microscopy, revealed a
twofold to fourfold reduction in the cross-sectional nuclear area and
in the overall cell area of treated KS Y-1 cells compared with
buffer-treated KS Y-1 cells. Thus, HAF in clinical-grade crude
preparations of hCG induces prototypical early cell morphology changes
of apoptosis.
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Because preparations of hCG are known to stimulate the growth of
some cells and induce apoptosis in others, we anticipate that
preparations of hCG-stimulated proliferation-associated pathways in KS
cells preferentially operate in these cells to accomplish programmed
cell death (21-26). After treatment with clinical-grade crude
preparations of hCG (400 IU/mL) for 24 hours, extracts of KS Y-1 and KS
SLK cells were examined for proliferation-associated protein expression
by western blot analysis. While buffer-treated cells and cells with no
additional treatment contained undetectable levels of c-Myc, both KS
Y-1 and KS SLK cells when treated with preparations of hCG showed
markedly enhanced levels of c-Myc (Fig. 6, A). In
parallel studies, treatment of KS Y-1 cells with preparations of hCG
(400 IU/mL) induced c-Rel protein expression, indicating a coordinated
expression of at least two proto-oncogenes known to participate in the
same signaling pathway (23) (Fig. 6, B).
Additionally, KS Y-1 cell expression of RB protein did not show changes in the overall
levels of RB after treatment with preparations of hCG (data not shown).
However, there was a preferential reduction in the hyperphosphorylated
form (higher molecular weight of the two bands) of RB in KS Y-1 cells
(Fig. 6, B)
after treatment with preparations of hCG. The diminished
hyperphosphorylated levels of RB is consistent with accumulation of
cells in G0/G1 phase and this type of cell cycle
regulation is characteristic of certain apoptotic pathways
(27). Moreover, western blot analysis showed that treatment
with preparations of hCG lowered Bcl-2 levels (by ~50%), whereas
the levels of p53 remained unchanged (data not shown), as commonly
noted in programmed cell death induced by the nongenotoxic type of
stimuli (22). Thus, these data indicate HAF ultimately induces
apoptosis following expression of proliferation-associated proteins as
observed with other mitogen-triggered apoptosis.
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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 susceptible to
Fas-mediated cell killing by treating cells with anti-Fas antibody
(0-100 µL 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 apoptosis involves mechanisms independent of Fas
receptor stimulation.
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DISCUSSION |
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The newly described apoptotic effect attributed to HAF resembles the apoptosis pathway best followed by a number of growth factors related to programmed cell death. In various cell systems, extracellular ligands and hormones that stimulate cell growth pathways can also, under the restricted conditions, induce apoptosis. Because preparations of hCG can induce proliferation and/or induce c-Myc in some normal and neoplastic cells (26,29), it was anticipated to precipitate apoptosis, perhaps by diverting an activated cell proliferation pathway to a cell death end point (23-25). Here, HAF-induced c-Myc and c-Rel expression, as well as induced c-Myc expression in transfectants, was followed by KS cell apoptosis, suggesting participation of these transcription factors in HAF-induced cell death. Preparations of hCG-stimulated c-Myc and c-Rel concur with results from other laboratories showing regulation of AP-1 complex formation in HAF-induced apoptosis (28,30). Even though c-Myc-related apoptosis has extensively been described in cell culture, a study (25) implicates c-Myc in apoptosis as a mechanism in cancer regression in vivo.
The identity of HAF is unknown. Fractions of preparations of hCG
containing HAF activity are distinct from hCG, its subunits, or nick
forms and is characterized as a protein by denaturing and protein
digest experiments (16). Others (31) have found
ribonuclease isolated from preparations of hCG to contain anti-KS
activity; however, the purified fractions with HAF prepared in our
laboratory lack ribonuclease. Fractions containing HAF-induced
apoptosis of KS cells were not blocked by antiribonuclease antibodies.
Interestingly, hCG is susceptible to proteolytic cleavage in
vivo and can potentially produce peptides that activate receptors.
The ß subunit of hCG can be potentially cleaved to form hCGß AA
37-57. Synthetic forms of this peptide form amphipathic helical
structures that can mimic the hCG heterodimer in activating its cognate
receptor, albeit at several-fold higher concentrations (32).
Since HAF activity is found in urine in early pregnancy (16), its presence may be linked to the substantially lower rates of AIDS-KS in women versus men (33). HIV-1-infected women have lower rates of HHV8 infection and KS compared with HIV-1-infected men. Even though nonpregnant women already exhibit lower rates of KS, one would predict that pregnancy via HAF would offer enhanced protection against development of KS or HHV8 infection. However, a single retrospective study by Rabkin et al. (34) in a small number of HIV-1-infected African women found that pregnancy was not associated with lower KS rates. For other types of cancers, such as mammary cancer in humans and rats, the pregnancy state does confer subsequent lower rates of tumor occurrence. During pregnancy, breast tissue undergoes differentiation that renders breast tissue cells resistant to transformation (35). In contrast, the HAF effect we observed with KS cells and preparations of hCG is primarily an apoptotic mechanism. Although stimulated cell differentiation can subsequently include apoptosis in some cells (36) and a transient cell differentiation effect in our studies cannot be ruled out, the overwhelming evidence suggests that HAF induces an immediate and primarily apoptotic effect. Indeed, apoptosis appears to be the basis for the demonstrated high remission rates (>80%) of intralesional preparations of hCG in a phase II trial of human KS (14) as well as high remission rates of the more advanced and resistant visceral KS in response to systemic treatments with preparations of hCG (15). The apoptosis activity of preparations of hCG indicates that a human-derived factor, HAF, is directly involved in KS cell death and holds promises for biologic-based anticancer therapy and further suggests the existence of apoptosis regulation that has previously been unappreciated.
Supported in part by a research supplement to R01AI38192-02S1 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (F. Samaniego).
We thank Anna M. Mazzuca and Stacy Cline for their editorial assistance.
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Manuscript received August 28, 1998; revised November 13, 1998; accepted November 18, 1998.
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