cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle.

Abstract

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G1 phase of the cell cycle instead of G0. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G0 phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G1, 12% were in S and 37% in G2 phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P < 0.01) [3H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P < 0.01) [3H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G1 phase of the cycle to enhance IGF-I effects in cell proliferation.

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Cell ProliJ: I995,28, 12 I - 1 36
CAMP and PMA enhance the effects of IGF-I in the
proliferation of endometrial adenocarcinoma cell line HEC- 1 -A
by acting at the G, phase of the cell cycle
F. Talavera, C. Bergman, M. L. Pearl, P. Connor, J. A. Roberts and
K. M. J. Menon
Department oJObstetrits and Gynecologl: Universiy oJ Michigun Medical Center, Ann Arbor, Michigan, USA
(lieceiverl 18 October 1094; rcwisiori ucceptecl23 Juniiury 1905)
Abstract. The present study was undertaken to determine whether endometrial cancer
cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein
kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G, phase of
the cell cycle instead of Go. Immunofluorescence staining of HEC-1-A cells using the
proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric
analysis determined that HEC-1-A cells do not enter the G,, phase of the cell cycle when
incubated in a serum-free medium. Approximately 5 1% of the cells were in G,, 12%
were in S and 37% in GI phase of the cell cycle prior to treatment. Forskolin and
phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and
protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased
( P < 0.01) [3HH]-thymidine incorporation in a dose and time dependent manner. The
interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated
with forskolin or PMA followed by incubation with IFG-I incorporated significantly
more (P<0.01) [3HH]-thymidine into DNA than controls or any treatment alone. It is
concluded that forskolin and, to a lesser extent, PMA exert their effect at the G, phase of
the cycle to enhance IGF-I effects in cell proliferation.
Insulin-like growth factor-I (IGF-I) is known to elicit a mitogenic response in vitro and in vivo in a
variety of normal and neoplastic cells (Sell et a/. 1993, Pietrzkowski et ul. 1992, Dickson ef a/.
1987, Tricoli eta/. 1986). Mitogenesis, however, is a multistep process where a competence signal
stimulates the cells to traverse the G,,/G, phase of the cell cycle, and progression factors such as
IGF-I then commit the cell to DNA synthesis. For example, studies performed with mouse
embryo have shown that the EGF receptor requires the presence of a functional IGF-I receptor
for its mitogenic and transforming activities (Coppola et ul. 1994). In addition, studies with Balb
C/3T3 cells, have shown that quiescent fibroblasts do not respond mitogenically to EGF unless
they have been pretreated with cAMP analogs (Olashaw et a/. 1988). Similarly, in FRTL-5 cells, a
line of nontransformed rat thyroid cells, the effects of IGF-I on [ 'HI-thymidine incorporation have
been shown to be significantly enhanced by prior exposure to cyclic AMP (Tramontano et a/.
Correspondence: Dr K. M. J. Menon, Dept. of Ob/Gyn, 1500 E. Medical Center Drive, L1221 Women's
Hospital, University of Michigan Medical Center, Ann Arbor, MI 48 109-0278, USA,
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F. 'lirluvem et al.
1988). However, cells from primary carcinomas, as well as established cancer cell lines, differ
from normal cells, in the sense that cancer cells arrest in the GI phase of the cell cycle instead of
G,, during density inhibition or serum deprivation (Tay et al. 199 1). Therefore, factors such as
CAMP or protein kinase C which are known to play a competence role in various cell systems.
would not act in cancer cells to recruit them from the G,, phase into the cell cycle but instead
enhance the mitogenic activity of growth factor stimulated cells by acting at the GI phase of thc
cell cycle.
We have been studying the growth regulation of transformed cell line HEC-I-A which is
derivcd from endometrial adenocarcinoma (Pearl ef al. 1993). This cell line possesses type 1 ICF
receptor (Perkonen et al. 199 1 ) and IGF-1 stimulates both [3H]-thymidine incorporation and cell
growth as evidenced by an increase in cell number (Talavera et ul. 1992). Furthcrrnore, trcatment
of this cell line with PMA and forskolin also stimulates mitogenesis, suggesting that protein kinasc
C and cyclic AMP mediated cellular mechanisms might trigger the growth of these transformed
cells (Talavera el ul. 1992). The purpose of the present study was to examine whether protein
kinase C and cyclic AMP would act at the G, phase of the cell cycle to further enhance IGF-l
effects in the mitogenic activity of endometrial adenocarcinoma cell line HEC-1 -A.
MATERlALS AND METHODS
Recombinant IGF-l was a generous gift from Eli Lilly Research Laboratories (Indianapolis, IN,
USA). Fatty acid free bovine serum albumin (BSA), fetal bovine serum (FBS), phorbol 12-
niyristate 1 3-acetate (PMA), forskolin, 8-bromo CAMP, and 1 oleoyl-2-acetyl glycerol (OAG)
were obtained from Sigma Chemical (St Louis, MO, USA). H-stain (Hoechst, 33258 dye) was
purchased from Aldrich (Milwaukee, WI, USA). Electrophoresis reagents and trypsin/EDTA
solution containing 0.25% trypsin and 0.02% EDTA, were obtained from BioRad (Richmond,
CA, USA).
Cell cultures
Human endometrial cancer cell line HEC- I-A (ATCC # HTB-112 batch # F-6720) was
purchased from the American Type Culturc Collection. The cells were maintained in DME/F 12
mixture (Sigma D2906) supplemented with 10'% fetal bovine serum, 50 pg/ml gentamycin, and
2 U/ml nystatin. The cells were cultured in 75 cm? tissue culture flasks (Corning Glass Works.
Corning, NY, USA) and maintained at 37°C in a water saturated air with 5% CO?. For
subculturing, the medium was removed and replaced with fresh buffer containing 0.25'%, trypsin
and 0.02'%, EDTA solution. After 2 min, the solution was removed and the flasks maintained ;it
37°C for approximately I 0 niin to allow the cells to detach. The cclls were suspended in fresh
medium and centrifuged at 1 SO x g for 10 min. The supernatant was discarded, and the cell pellet
resuspended in fresh medium. The resuspended cells were cultured in 24 well plates and incubated
in serum-free DME medium (0.3% BSA) for 24 h to allow for cell attachment. Following this
incubation, the cells were trcated immcdiately as described in the figures. Given the aggressive
growth of these cells in serum-free medium. possibly due to autocrine growth factors being
secreted by the cells, initial results were often inconsistent regarding the magnitude of their
response to growth promoting agents. However, we have found that a consistent response to
growth factor stimulation is observed when the cells arc washed extensively (three times) with
serum-free medium prior to treatment and when the cclls are treated 24 h following their initial
plating in the absence of serum. These conditions were used for all incubations in our studies.
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CAMP arid PMA act at the G, phase
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Immunofluorescence staining of HEC- 1 -A cells
The endometrial HEC- 1-A cells were removed from the wells with fresh buffer containing 0.25%
trypsin and 0.02% EDTA solution, as described above, for subculturing and washed in PBS. The
cell pellet was resuspended in 500 pl of a solution containing 1% paraformaldehyde, vortexed
and incubated for 20min. Following a wash with PBS, the cells were then mounted on a
microslide for fluorescent microscopic observation (Menge et ul. 1993). The fixed cells were
incubated with 200 pl containing 20 pg/ml of mouse proliferating cell nuclear monoclonal
antibody (PCNA IgG,,,: Oncogene Science, Uniondale, NY, USA). Negative controls were
incubated in the presence of mouse IgGzil. The cells were then washed with 2ml of PBS and
incubated with 200 p1 of a second antibody (anti IgG,,) labelled with fluorescein isothiocyanate
(FITC: Dako, Carpinteria. CA, USA). Slides were examined using a Fluorescence microscope
(Carl Zeiss, Germany).
To analyse for the presence of the cell cycle dependent protein Ki-67, the
immunofluorescence analysis was carried out as described by Coltrera and Cown ( 199 1 ). In brief,
the cells were fixed using absolute methanol for 30min. The fixed cells were incubated with
200 pl of mouse Ki-67 antibody (Dako Corporation, Santa Barbara, CA, USA) at 1 : 25 dilution.
The cells were then washed with 2 ml of PBS and incubated with 200 pl of Fluorescin-conjugated
(FITC; 1 : 100) goat anti-mouse anti-lgG (Fc specific), purchased from Sigma Chemical (St. Louis,
MO, USA). The antibodies were diluted with PBS containing 1°L1 BSA.
Flow cytometry and cell cycle analysis
Cells were plated at a density of 50 000 cells/well in DME, incubated with bromodeoxyuridine
(BrdUrd; 10 PM) for 30 min prior to harvesting and processed for indirect immunofluorescence
staining as described by the manufacturer (Beckton Dickinson lmmunocytometry Systems, San
Jose, CA, USA). Processed cells were analysed using an Elite Flowcytometer (Coulter
Corporation, Hialeah, FL, USA) fitted with an Argon Ion Laser adjusted to emit 15 mW at
488 nm. Green fluorescence was measured through a BP525 nm filter. Propidium iodide red
fluorescence yielded cell cycle distributions unaffected by BrdUrd incorporation and was
measured through a BP630 nm filter. The bivariate fluorescent distributions were displayed in
dot blots (64 x 64 channel array). Each analysis was carried out on at least 10 000 events and an
analysis region was placed on the dual parameter histogram of the isotype control, so that 5% of
the cells in the control sample were located in the region. Any sample giving a signal of greater
than 5% above the control was considered positive. Compartmentalization of the cell cycle was
analysed on an Epics Elite Workstation (Coulter Corporation).
Thymidine incorporation assay
Thymidine incorporation studies were performed in 24 well plates (Imai el al. 1982). Cells were
plated at a density of 50000 cells/well in DME and treated for 24 h intervals, unless specified
otherwise. Four hours prior to completion, 1 pCi [-'H]-thymidine was added to each well. Cells
were then washed with ice cold PBS and incubated with TCA (5"h) to remove the acid soluble
['HI-thymidine pool. Cells werc washed again with PBS and dissolved in 500 pl NaOH (0.2 M) to
recover the remaining incorporated ['HI-thymidine. This was transferred to scintillation vials
containing 10 ml Biosafe 11. After neutralization with 0.2 N HCI, the radioactivity was determined
using a beta particle counter.
Cell growth assay
HEC- 1 -A cells (50 000 cells/well) were plated in quadruplicate in 24 well plates containing
500 pl phenol red-free DME/F 12 medium containing 0.3"/0 BSA in the absence or presence of
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F. Tuluveru et al.
various treatments. Following the incubations, the cells were harvested with 500 pI trypsin/EDTA
solution, as described for cell cultures. The trypsin was neutralized with 500 pI fresh medium.
The cell number was quantitated in four aliquots from each replicate using a hemocytomcter.
IGF-I binding assay
IGF-1 iodination and binding assays were carried out as described previously (Talavera ct al.
1990) with slight modifications. Briefly, cells were incubated with treatments as described in the
figures. At the end of the treatment period, cells were washed and incubated with medium
containing '2sI-IGF-I in the absence or presence of a 200-fold excess of unlabelled ligand.
Incubations were performed at 4°C for 16 h. Unbound growth factor was removed with ice-cold
growth medium wash. Cells were then lysed with 0.5 N sodium hydroxide and incubated at 37°C
for 30 min. The radioactivity was quantitated in a gamma counter. Specific binding was
determined by subtracting the nonspecific binding from total binding.
DNAassay
HEC-1-A cells were cultured as described for the growth assay. DNA was analysed by the
procedure of Labarca & Paigen (1982). The cells were solubilized with 0.25 ml of 0.5 N sodium
hydroxide and incubated at 37°C for 1 h. High-salt DNA assay buffer (0.75 ml, 0.5 M sodium
phosphate, 2 M sodium chloride, pH 7.4) and bisbenzimidazole (32 pg/ml) were added and the pH
adjusted to 7.2-7.4 with 0.5 N hydrochloric acid. DNA standard5 were prepared using calf thymus
DNA. Fluorescence was measured at wavelengths set at 356 nM (excitation) and 458 n M
(emission).
Statistical analysis
The data represent the mean +SEM of the number of determination analysed by ANOVA and a
Student's I-test to compare differences among treatment groups. Each experiment was repeated
at least four times.
RESULTS
Expression of PCNA on HEC-I-A cells and flow cytometric analysis
Initial studies were performed to determine the percentage of cells that were not in the GI, phase
of the cell cycle through immunohistochemical studies using the monoclonal antibody to PCNA.
This antibody is not taken up by cells that are either in GI, or M phase of the cell cycle. Figure 1
shows the cells observed under fluorescent light. The percentage of cells staining positive for
PCNA was 100%, suggesting that the cells were not in G,, phase of the cell cycle when trcated.
Negative controls that were incubated in the presence of mouse IgGza did not stain (data not
shown). Some cells exhibited more fluorescent light than others which is consistent with cells that
are found at different phases of the cell cycle.
To further confirm our observations that these cells do not exit to the G,, phase when
incubated in the absence of serum, the cells were analysed for the presence of Ki-67 protein
which is known to be expressed throughout the cell cycle but not in the GI, phase. In order to
examine this, the cells were either plated at a density o f 50000 ml- ' (low density), or grown to a
density arrested state (high density) and incubated in the absence of serum (0.3% BSA) for 24 h
prior to the analysis. Figure 2a shows the staining of cells plated at low density. Figure 2b shows
the staining of cells that were density arrested prior to the analysis. Some cells exhibited more
fluorescence than others. However, the percentage of cells staining positive for Ki-67 was 10C1"/,,
for both low and high density plating suggesting that HEC-I-A cells do not exit to GI, when they
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Figure 1. PCNA expression in HEC-I -A cells as identified by indirect immunofluorescence. Cells were
grown to contluency and subcultured into 24 well plates for 24 h in the absence of serum. Following this
incubation, the cells were harvested and analysed through immunostaining as debcribed in the Materials and
Methods section.
Light
Fluorescence
Figure 2. Ki-67 expression in HEC-I-A cells as identified by indirect immunofluorescence. a Cells were
plated at 50000/ml and incubated in the absence of serum for 24h. b Cells were density arrested and
incubated in the absence of serum for 24 h. Following this incubations, the cells wcre harvested and analysed
through immunostaining as described in the Materials and Methods section.
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