Radiation-stimulated ERK1/2 and JNK1/2 signaling can promote cell cycle progression in human colon cancer cells.

Abstract

The abilities of mutated active K-RAS and H-RAS proteins, in an isogenic human carcinoma cell system, to modulate the activity of signaling pathways and cell cycle progression following exposure to ionizing radiation is largely unknown. Loss of K-RAS D13 expression in parental HCT116 colorectal carcinoma cells blunted basal ERK1/2, AKT and JNK1/2 activity by -70%. P38 activity was not detected. Deletion of the allele to express activated K-RAS nearly abolished radiation-induced activation of all signaling pathways. Expression of H-RAS V12 in HCT116 cells lacking an activated RAS molecule (H-RAS V12 cells) restored basal ERK1/2 and AKT activity to that observed in parental cells, but did not restore or alter basal JNK1/2 and p38 activity. In parental cells radiation (1 Gy) caused stronger ERK1/2 pathway activation compared to that of the PI3K/AKT pathway. In H-RAS V12 cells radiation caused stronger PI3K/AKT pathway activation compared to that of the ERK1/2 pathway. Radiation (1 Gy) promoted S phase entry in parental HCT116 cells within 24h, but not in either HCT116 cells lacking K-RAS D13 expression or in H-RAS V12 cells. In parental cells radiation-stimulated S phase entry correlated with ERK1/2-, JNK1/2- and PI3K-dependent increased expression of cyclin D1 and cyclin A, and to a lesser extent cyclin E, 6-24 h after exposure. Cyclin A and cyclin D1 expression were not increased by radiation in cells lacking K-RAS D13 expression or in H-RAS V12 cells. Radiation (1 Gy) modestly enhanced expression of p53, hMDM2 and p21 in parental cells 2-6 h after exposure, which was abolished in cells lacking K-RAS D13 expression. Introduction of H-RAS V12 into cells lacking mutant active RAS partially restored radiation-induced expression of p21 and p53, and enhanced the induction of hMDM2 beyond that observed in parental cells. Collectively, our findings argue that the coordinated activation of multiple signaling pathways, in particular ERK1/2 and JNK1/2, by radiation is required to elevate the expression of G1 and S phase cyclin proteins and to promote S phase entry in human colon carcinoma cells expressing wild type p53. In HCT116 cells H-RAS V12 promotes hMDM2 expression after radiation exposure which correlates with reduced p53 expression and increased cell survival.

Full text

Page 1

©2005 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
and a Leukemia Society of America grant 6405-97.
A portion of Dr. Yacoub’s funding is from the
Department of Radiation Oncology, Virginia
Commonwealth University. The Massey Cancer
Center Flow cytometry core laboratory was sup-
ported in part by NIH grant P30 CA16059.
P.D. would like to thank Dr. E. Bernhard
(University of Pennsylvania) for useful discussions
during the initiation of these studies.
[Cell Cycle 4:3, 456-464; March 2005]; ©2005 Landes Bioscience
456
Cell Cycle
2004; Vol. 4 Issue 3
Rubén W. Carón 1,4
Adly Yacoub1
Clint Mitchell1
Xiaoyu Zhu1
Young Hong1
Takehiko Sasazuki3
Senji Shirasawa3
Michael P . Hagan1
Steven Grant2
Paul Dent1,*
1Department of Radiation Oncology; 2Department of Hematology/Oncology;
Virginia Commonwealth University, Richmond, Virginia USA
3Department of Pathology, International Medical Center of Japan; Shinjuku-ku,
Tokyo, Japan
4IMBECU-CONICET; Mendoza, Argentina
*Correspondence to: Paul Dent, Ph.D.; Department of Radiation Oncology;
Medical College of Virginia; Virginia Commonwealth University; 401 College
Street, Richmond, Virginia 23298-0058, USA; Tel.: 804.628.0861; Fax:
804.828.6042; Email: pdent@hsc.vcu.edu
Received 09/17/04; Accepted 09/28/04
Previously published online as a Cell CycleE-publication:
http://www.landesbioscience.com/journals/cc/abstract.php?id=1249
KEY WORDS
radiation, RAS, signaling, cell cycle
ABBREVIATIONS
MAPK
PI3K
ERK
JNK
hMDM2
mitogen activated protein kinase
phosphatidylinositol 3-kinase
extracellular regulated kinase
c-Jun NH2-terminal kinase
human form of the murine double
minute protein
ACKNOWLEDGEMENTS
This work was funded; to P.D. from PHS grant
(R01-CA88906, P01-CA72955, R01-DK52825),
Department of Defense Awards (BC980148,
BC020338); to S.G. from PHS grants
(P01-CA72955; R01-CA63753; R01-CA77141)
Report
Radiation-Stimulated ERK1/2 and JNK1/2 Signaling Can Promote Cell
Cycle Progression in Human Colon Cancer Cells
ABSTRACT
The abilities of mutated active K-RAS and H-RAS proteins, in an isogenic human carci-
noma cell system, to modulate the activity of signaling pathways and cell cycle progression
following exposure to ionizing radiation is largely unknown. Loss of K-RAS D13 expression
in parental HCT116 colorectal carcinoma cells blunted basal ERK1/2, AKT and JNK1/2
activity by ~70%. P38 activity was not detected. Deletion of the allele to express activated
K-RAS nearly abolished radiation-induced activation of all signaling pathways.
Expression of H-RAS V12 in HCT116 cells lacking an activated RAS molecule (H-RAS
V12 cells) restored basal ERK1/2 and AKT activity to that observed in parental cells, but
did not restore or alter basal JNK1/2 and p38 activity. In parental cells radiation (1 Gy)
caused stronger ERK1/2 pathway activation compared to that of the PI3K/AKT pathway.
In H-RAS V12 cells radiation caused stronger PI3K/AKT pathway activation compared to
that of the ERK1/2 pathway. Radiation (1 Gy) promoted S phase entry in parental
HCT116 cells within 24h, but not in either HCT116 cells lacking K-RAS D13 expression
or in H-RAS V12 cells. In parental cells radiation-stimulated S phase entry correlated with
ERK1/2-, JNK1/2- and PI3K-dependent increased expression of cyclin D1 and cyclin A,
and to a lesser extent cyclin E, 6–24 h after exposure. Cyclin A and cyclin D1 expres-
sion were not increased by radiation in cells lacking K-RAS D13 expression or in H-RAS
V12 cells. Radiation (1 Gy) modestly enhanced expression of p53, hMDM2 and p21 in
parental cells 2-6h after exposure, which was abolished in cells lacking K-RAS D13
expression. Introduction of H-RAS V12 into cells lacking mutant active RAS partially
restored radiation-induced expression of p21 and p53, and enhanced the induction of
hMDM2 beyond that observed in parental cells. Collectively, our findings argue that the
coordinated activation of multiple signaling pathways, in particular ERK1/2 and
JNK1/2, by radiation is required to elevate the expression of G1and S phase cyclin pro-
teins and to promote S phase entry in human colon carcinoma cells expressing wild type
p53. In HCT116 cells H-RAS V12 promotes hMDM2 expression after radiation exposure
which correlates with reduced p53 expression and increased cell survival.
INTRODUCTION
Ionizing radiation is used as a primary treatment for many types of carcinoma. While
it has been appreciated for many years that radiation causes cell death, it has only recently
become accepted that radiation has some potential to enhance proliferation in the surviving
fraction of cells.1,2We and others have discovered that exposure of carcinoma cells to low
radiation doses causes an initial early activation of growth factor receptors in the plasma
membrane, followed by secondary receptor activation that is dependent upon autocrine
growth factors.3,4Receptor activation enhances the activities of RAS family molecules that
signal to cause activation of multiple intracellular signal transduction pathways. Secondary
activation of intracellular signal transduction pathways by growth factors and radiation has
been correlated to altered expression of cell cycle regulatory proteins and may, under certain
circumstances, promote cell proliferation.5,6
Growth factors interact with plasma membrane receptors, which transduce signals
through the membrane to its inner leaflet.7-14Growth factor signals, via guanine
nucleotide exchange factors, can increase the amount of GTP bound to membrane-asso-
ciated GTP binding proteins, including RAS.15,10There are 3 widely recognized isoforms
of RAS: Harvey (H), Kirsten (K) and Neuroblastoma (N).16GTP-RAS can interact with
multiple downstream effector molecules including the Raf-1 protein kinase and the
phosphatidyl inositol 3-kinase (PI3K) lipid kinase. Receptor-stimulated guanine
nucleotide exchange of ‘RAS’ to the GTP-bound form permits Raf-1 and P110 PI3K to

Page 2

associate with ‘RAS’, resulting in kinase translocation to the plasma
membrane environment where activation of these kinases, via com-
plex mechanisms, takes place. RAS contains a GTPase activity that
converts bound GTP to GDP resulting in inactivation of the RAS
molecule. PI3K enzymes are also translocated to the plasma mem-
brane environment via the P85 SH2 domain interaction with phos-
phorylated tyrosine residues on adaptor proteins and growth factor
receptors, e.g., GAB2, IRS-1, ERBB3.17
Mutation of RAS results in a loss of GTPase activity, generating
a constitutively active RAS molecule that can lead to elevated activ-
ity within downstream signaling pathways. Approximately one third
of human cancers have RAS mutations, primarily the K-RAS isoform,
that also leads to a radio-protected phenotype.15,18Of note is that
some studies suggest that K-RAS and H-RAS have different but
over-lapping signaling specificities to downstream pathways as
judged by in vitro cell based studies and in animal knock-out models:
thus mutant K-RAS is thought to preferentially activate the Raf-1 /
extracellular regulated kinase (ERK1/2) pathway, whereas mutant
H-RAS is believed to preferentially activate the PI3K/AKT pathway.
19-21It has been argued that ERK1/2 and PI3K signaling down-
stream of K-RAS and H-RAS, respectively, can in turn control cell
growth and cell survival following exposure to multiple growth factors,
e.g., EGF.3,8,9
Recent studies in rodent fibroblasts have argued that H-RAS and
K-RAS can differentially alter radiosensitivity, with mutated active
K-RAS promoting radiation-sensitization, which was linked to acti-
vation of the p38 MAPK pathway.22However the roles of different
RAS isoforms and the signaling pathways utilized by radiation to
alter cell growth after exposure and radiosensitivity of isogenic
human carcinoma cells are unknown. Our findings argue that the
coordinated activation of multiple signaling pathways, in particular
ERK1/2 and JNK1/2, by radiation is required to elevate the expression
of G1and S phase cyclin proteins and to promote S phase entry in
human colon carcinoma cells expressing wild type p53. In HCT116
cells H-RAS V12 promotes hMDM2 expression after radiation
exposure which correlates with reduced p53 expression and
increased cell survival.
MATERIALS AND METHODS
Materials. Anti-Phospho-ERK1/2 (sc-7383), anti-total-ERK2 (sc-154),
anti-beta-actin (sc-8432) antibodies and protein A/G conjugated agarose
(sc-2003) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-Phospho-JNK1/2 (9255), anti-Phospho-Ser473-AKT (9271), anti-
Phospho-Thr308-AKT (9275), anti-total-AKT (9272), and Anti-
Phospho-p38 (9211) antibodies were from Cell Signaling Technology
(Beverly, MA). Antibodies to detect total RAS (OP24), H-RAS (OP23) and
mutant K-RAS (OP38) were from Oncogene Research Products
(Cambridge, MA). Antibodies anti-PI3K p85 (06-496) and anti-PI3K
p110α (06-567) were from Upstate Biotechnology (Lake Placid, NY).
Oligonucleotides for semi quantitative PCR were synthesized by the VCU
oligonucleotide core facility and were based on published sequences (sense
K-RAS: GAG AGA GGC CTG CTG AAA ATG, antisense K-RAS: TTC
TGA GGA CCG ACA CAC TTT, sense H-RAS: GCA GGC CCC TGA
GGA GCG ATG, antisense H-RAS: TCA GGG AGA GCA CAC ACT
TGC AGC, sense N-RAS: GGT TCT TGC TGG TGT GAA ATG, anti-
sense N-RAS: ACT TTA AAA GTA ATC TTG TTA CAT, sense β-actin:
GAA CCC taa ggc caa ccg tga aaa gat gac, antisense β-actin: TGA TCT
TCA TGG TGC TAG GAG CCA GAG CAG). The MEK1/2 inhibitors
U0126 and PD184352, the JNK1/2/3 inhibitor SP600125 and the PI3K
inhibitor LY294002 were from Calbiochem (San Diego, CA). Other reagents
were as in references 23–25.
Methods
Generation of HCT116 cell lines without mutant K-RAS and expressing
mutant H-RAS. HCT116 mutant K-RAS-deleted cells were generated by
homologous deletion of the mutant K-RAS allele as described.26,27A plas-
mid to express mutant active H-RAS (H-RAS V12) was kindly provided by
the laboratory of Dr. M. Wigler (Cold Spring Harbor, NY). A plasmid to
express dominant negative AKT (AKT1-S473A/T308A mutant) was kind-
ly provided by Dr. R.A. Roth (Stanford University, Palo Alto, CA).
HCT116 cell lines were transfected by electroporation at 600 V for 60 msec
using a Multiporator Eppendorff (Hamburg, Germany) with control plas-
mids (“C2” cells) or plasmids to express H-RAS V12 (“C10” and “C3”
cells). Pools of transfected cells were obtained by puromycin selection and
individual colonies isolated and then characterized.
Culture of HCT116 cell lines. Asynchronous carcinoma cells were cul-
tured in DMEM media supplemented with 10% (v/v) fetal calf serum at
37˚C in 95% (v/v) air / 5% (v/v) CO2. Cells were plated at a density 3 x 103
cells/cm2plate area and all cells were plated from log phase cultures. For
radiation-induced activations of protein kinases, cells were cultured for four
days in this media, and for 24 h prior to irradiation were cultured in serum
free DMEM medium. For colony formation assays, cells were plated at low
density (250–1,000 cells per dish) and 24 h after plating, for the 24 h prior
to irradiation, were cultured in serum free DMEM medium. Cells were irra-
diated (1–4 Gy) or as indicated in the text: media was replaced with serum
containing media 24h after radiation exposure. Ten-14 days after exposure,
plates were washed in PBS, fixed with methanol and stained with a filtered
solution of crystal violet (5% w/v). After washing with tap water, the
colonies were counted both manually (by eye) and digitally using a
ColCount TM plate reader. Data presented is the arithmetic mean (± SEM)
from both counting methods from multiple studies.
Exposure of cells to ionizing radiation and cell homogenization. Cells
were cultured as described above. As indicated, 1h prior to irradiation cells
were treated with either vehicle (DMSO), U1026 (1 µM), PD184352 (2
µM), SP600125 (1 µM) or LY294002 (1 µM). Treatment was from a 100
mM stock solution and the maximal concentration of vehicle (DMSO) in
media was 0.01% (v/v). Cells were irradiated using a 60Co source at dose
rate of 1.8 Gy/min. Cells were maintained at 37˚C throughout the experi-
ment except during the irradiation itself. Zero time is designated as the time
point at which exposure to radiation ceased. After radiation-treatment cells
were incubated for specified times followed by aspiration of media and
immediately homogenized in 1 ml SDS PAGE lysis buffer [5% (w/v) SDS,
40% (v/v) glycerol, 250 mM Tris-HCl, 10% (v/v) 2-mercaptoethanol].
Homogenates were sonicated, boiled for 10 min, the protein concentration
determined by Bradford assay (Coomassie Protein Assay Kit, Pierce
Biotechnology, Rockford, IL) and stored frozen (-20˚C) prior to use.
SDS poly-acrylamide gel electrophoresis (SDS PAGE) and western
blotting. Depending on the protein to be studied, a volume of homogenate
containing 10, 20 or 40 µg of total protein was loaded in 12% (w/v) acry-
lamide gels and subjected to SDS PAGE. Gels were transferred to nitrocel-
lulose and western blotted using specific antibodies. Blots were developed
using Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer
Life Sciences, Boston, MA) and Kodak x-ray film, or using an Odyssey
Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) which has
3-log detection sensitivity over conventional E.C.L. immunoblotting. This
machine utilizes dye-conjugated secondary antibodies that fluoresce in
infrared light as a detection system rather than an enzymatic chemilumines-
cent reaction. Secondary antibodies are available that fluoresce red or green,
which are detected on two separate channels in/by the machine (Molecular
Probes, Rockland ImmunoChem.). Thus immunoblots to detect multiple
proteins of different masses can be digitally scanned/probed on the same
piece of nitrocellulose at the same time. Densitometric analysis for E.C.L.
immunoblots and RT-PCR analyses was performed using a Fluorochem
8800 Image System and the respective software (Alpha Innotech
Corporation, San Leandro, CA) and band densities were normalized to that
of β-actin in the same sample and expressed as a percentage of the respective
control in each experiment as indicated in the legends to figures. Blots were
digitally scanned using Adobe Photoshop 7, their color removed, and
Figures created in Microsoft PowerPoint.
www.landesbioscience.com
Cell Cycle
457
Radiation, RAS and Cell Cycle Regulation

Page 3

PCR analysis of mRNA levels. Cells were plated in 10% FCS-DMEM
and 96 h after plating the media replaced by either serum containing media
or media without serum. Twenty four hours after the media change, cells
were homogenized in buffer containing guanidine isothiocyanate for the
extraction of total RNA with a commercial kit (Rneasy) following the direc-
tion of the manufacturer (Qiagen, Valencia, CA). RNA yield and purity were
determined following measurement of absorbance at 260 and 280 nm. The
reverse transcriptase-polymerase chain reaction (RT-PCR) was performed
using a commercial kit (Quiagen OneStep RT-PCR) and 1 µg of RNA in a
final volume of 25 µl and following the instructions of the manufacturer
(Qiagen, Valencia, CA). To determine the expression of H-, K- and N-RAS,
and β-actin, epiregulin, heregulin, TGFα, HB-EGF, EGF, ERBB1, ERBB2,
ERBB3 and ERBB4, the respective primers described in Materials at a final
concentration of 0.6 µM were used. Both, the reverse transcription and the
DNA synthesis were performed in the same tube in a Peltier Thermal Cycler
200 (MJ Research Inc., Boston, MA). The reverse transcriptase reaction was
performed at 50˚C for 30 min followed for a step of activation of the DNA
polymerase and inactivation of the reverse trancriptase at 95˚C for 15 min
and 25 cycles of denaturalization at 95˚C for 1 min, annealing at 50˚C for
1 min, and extension at 72˚C for 1 min and one cycle of 10 min at 72˚C.
All PCR products were analyzed by gel electrophoresis through a 1% agarose
gel containing ethidium bromide at 100 V for about 1 h in Tris-Borate-
EDTA buffer. Densitometric analysis was performed using a Fluorochem
8800 Image System and the respective software (Alpha Innotech
Corporation, San Leandro, CA) and band densities were normalized to that
of β-actin in the same sample and expressed as a percentage of the respective
control in each experiment as indicated in the legends to figure legends.
Cell cycle analysis: propidium iodide staining of cells. After plating (96
h), the medium was replaced by serum-free media and twenty four h later,
the cells were harvested by tryptic digestion and aliquots containing 1 x 106
cells were pelleted by centrifugation at 1500 rpm, for 5 min and resuspend-
ed in 1.5 ml of PBS and pelleted again. The pellet was suspended in 1.0 ml
of propidium iodide stain containing 3.8 mM sodium citrate, 7 U/ml RNAse
B (ICN, Irvine, CA), 0.1% Triton X-100 and 0.05 mg/ml propidium iodide
(Sigma, Saint Louis, MO) and incubated at 4˚C overnight. The suspension
of cells was filtered through a mesh of nylon number 40 (Small Parts Inc,
Miami Lakes, FL). Cells were analyzed with an Epics XL-MCL flow
cytometer (Beckman Coulter Inc.) and the data acquisition with Expo 32
ADC software. The analysis of the data was performed with Mod Fit LT 2.0
(Verity software).
Data analysis. Comparison of the effects of treatments was performed
using one-way analysis of variance and a two-tailed t-test. Differences with
a P-value of < 0.05 were considered statistically significant. Experiments
shown, except where indicated, are the means of multiple individual points
from multiple separate experiments (± SEM).
RESULTS
Generation and characterization of wild type/parental, mutant K-RAS
deleted and H-RAS V12 expressing HCT116 cells. To examine the relative
roles of mutant active K-RAS and mutant active H-RAS molecules in the
responses of isogenic tumor cells to radiation, we obtained previously
described HCT116 cell lines that had been genetically manipulated with the
allele expressing K-RAS D13 deleted by homologous recombination, while
leaving the other allele of wild type K-RAS intact (mutant K-RAS-deleted
cells, clone 2).26,27HCT116 cell lines were stably transfected with either a
vector control plasmid or a plasmid to express H-RAS V12 (H-RAS V12
cells, clones C10 and C3). Colonies were initially characterized and selected
for further study based on total RAS protein expression (data not shown).
Compared to parental HCT116 cells, and as would be predicted upon
losing function of one allele (of a gene), HCT116 mutant K-RAS-deleted
cells exhibited a ~50% reduction in the levels of K-RAS mRNA and protein
expression (Fig. 1A and B). Transfection of active H-RAS V12 into mutant
K-RAS deleted cells resulted in a ~80% increase in H-RAS protein levels
above wild type cells and a restoration of H-RAS mRNA levels to near those
found in wild type cells (Fig. 1A and B). N-RAS mRNA levels were approx-
imately 25% of those found for K-RAS, and the N-RAS isoform was not
reproducibly detected by protein immunoblotting in any HCT116 cell line
(data not shown).
Differential regulation of signaling pathway activities by mutant
K-RAS and mutant H-RAS. RAS molecules have been proposed to differ-
entially regulate the activities of multiple downstream pathways comparing
kinase activities in nonisogenic cell types, in particular K-RAS regulating the
Raf-1/MEK/ERK1/2 pathway and H-RAS controlling the PI3K/AKT/
GSK3 pathway. Radiation resistance downstream of mutant K-RAS and
mutant H-RAS, also using nonisogenic cells, has generally been linked
predominantly to activation of the PI3K pathway.28,29
Loss of mutant K-RAS expression significantly reduced basal ERK1/2
and AKT activity in HCT116 cells (Fig. 2A and B). Transfection of mutant
K-RAS deleted cells with H-RAS V12 restored basal ERK1/2 and AKT
activity to those found in wild type cells. Loss of mutant K-RAS expression
almost abolished basal JNK1/2 activity which was not restored by expression
of H-RAS V12 (Fig. 2C). Identical data for ERK1/2, AKT and JNK1/2
were obtained using nontransfected mutant K-RAS deleted cells and in an
additional HCT116 clone expressing mutant H-RAS V12 (data not shown).
In contrast to contemporaneous studies in rodent fibroblasts transfected to
express mutated active H- and K-RAS proteins, 22basal p38 activity was not
detected in any of the HCT116 carcinoma cell lines examined (Fig. 2C).
K-RAS D13 promotes radiation-induced ERK1/2 pathway activation
in parental cells and H-RAS V12 promotes radiation-induced PI3K/ AKT
pathway activation H-RAS V12 (C10) cells. We next examined modulation
of signaling pathway activities over a 24 h time course following a 1 Gy radi-
ation exposure in transfected HCT116 cell lines. In contrast to findings in
rodent fibroblasts, p38 was not activated following a 1 Gy exposure in any
HCT116 cell type examined (Fig. 3A). In parental/wild type HCT116 cells,
JNK1/2 was activated in two phases, 5–30 min and 1–5 h after exposure,
however no activation of JNK1/2 was observed in either mutant
K-RAS-deleted cells or H-RAS V12 expressing cells (Fig. 3A, data not shown).
ERK1/2 and AKT were also activated in multiple phases by radiation in
HCT116 cell lines (Fig. 3B and C). In parental cells, ERK1/2 was activated
in three phases following radiation exposure. After exposure ERK1/2
Radiation, RAS and Cell Cycle Regulation
458Cell Cycle2005; Vol. 4 Issue 3
Figure 1. Expression of H-RAS, K-RAS and N-RAS in HCT116 cells trans-
fected with empty vector or a mutant H-RAS plasmid. (A) Levels of mRNA for
H-RAS, K-RAS and N-RAS in HCT116 cell clones. The mRNA levels of H-, K-,
N-RAS and β-actin were determined by RT-PCR using the specific primers as
described in Materials and Methods. Data are from a representative exper-
iment (n = 3–5), showing WT, C2 (mutant K-RAS deleted), C10 (mutant
K-RAS deleted expressing H-RAS V12) and C3 (mutant K-RAS deleted
expressing H-RAS V12). (B) Expression of K-RAS and H-RAS proteins deter-
mined by immunoblotting using RAS isoform specific antibodies. Data are
from a representative experiment (n = 3–5) showing WT, C2 (mutant K-RAS
deleted), C10 (mutant K-RAS deleted expressing H-RAS V12).
A
B

Page 4

phosphorylation was enhanced shortly following (5–30 min), several hours
after (1–4 h) and at times many hours later (6–24 h) (Fig. 3B). Compared
to wild type cells, the activation of ERK1/2 was significantly reduced 5–30
min after exposure in mutant K-RAS-deleted (C2) cells, and was abolished
thereafter (30 min to 24 h). In cells expressing H-RAS V12 (C10 cells) the
activation of ERK1/2 was enhanced to a greater extent than in wild type
cells shortly following exposure (5–30 min); however ERK1/2 activation
was only partially restored several hours after exposure (1–4 h). At later
times (6–24 h), ERK1/2 was not activated by radiation in H-RAS V12 cells.
In parental HCT116 cells, AKT was modestly activated shortly following
exposure (5–30 min), several hours after exposure (1–4 h) but was not acti-
vated at later times (6–24 h) (Fig. 3C). Activation of AKT was abolished in
mutant K-RAS-deleted cells. In cells expressing H-RAS V12, AKT was
strongly activated, which occurred in three phases, (5–30 min), (1–4 h) and
(6–24 h) after exposure. The increase in AKT activation in cells expressing
H-RAS V12 was significantly enhanced beyond the level observed for AKT
in parental/wild type cells. Similar data were obtained in one additional
clone expressing H-RAS V12 (not shown).
Low dose radiation promotes S phase entry in parental HCT116 cells,
but not in mutant K-RAS deleted (C2) cells or in H-RAS V12 (C10) cells.
As radiation activated ERK1/2 and AKT in both parental and H-RAS V12
HCT116 cell lines, we next investigated whether radiation modulated the
growth rate of HCT116 cell lines. In proliferating HCT116 cell lines, 24 h
after a 1 Gy radiation exposure, no significant alteration in total cell num-
bers of parental or H-RAS V12 cells was observed as judged in MTT assays
(data not shown). We next investigated whether radiation was capable of
stimulating cell cycle progression in HCT116 cell lines cultured in the
absence of serum. These cells have approximately 20% of the growth
rate/doubling time of cells cultured in the presence of 10% (v/v)
fetal calf serum. Cells were plated and treated in an identical
manner to those for protein kinase activity assays, exposed to
radiation, and the percentage of cells in G1, S and G2/M phases
determined 24 h after exposure. In parental cells, radiation
reduced the percentage of cells in G1phase and increased the
number of cells present in S phase, but did not alter cell numbers
in G2and M phases (Fig. 4A). In contrast, radiation did not alter
the G1/S/G2/M cell cycle distribution profiles of either mutant
K-RAS deleted (C2) cells or, more surprisingly, of H-RAS V12
(C10) cells.
Based on data showing that ERK1/2 and JNK1/2 were acti-
vated more potently by radiation in parental cells compared to
H-RAS V12 cells, and based on evidence that ERK1/2 and
JNK1/2 signaling promotes S phase entry in many cell types, we
next investigated in parental HCT116 cells whether inhibition of
MEK1/2 and/or JNK1/2 function blocked the reduction in G1
phase and the increase in S phase presented in Figure 4A.
Inhibition of MEK1/2 caused a large increase in the percentage of
cells present in G1phase, which was not altered following radiation
exposure (Fig. 4B). MEK1/2 inhibition reduced cell numbers in
S phase, which also was not changed after radiation exposure.
Inhibition of JNK1/2 signaling did not significantly alter the
www.landesbioscience.com
Cell Cycle
459
Radiation, RAS and Cell Cycle Regulation
Figure 2 (Above). Basal levels of P-ERK1/2, P-AKT S473 and P-JNK1/2 in
HCT116 clones. Phosphorylation (activity) of protein kinases was determined
by immunoblotting using specific antibodies for the phosphorylated forms of
(A), ERK1/2, (B), AKT S473 and (C). JNK1/2 and p38 in HCT116 cells
(WT), mutant K-RAS-deleted cells (C2) or mutant K-RAS-deleted cells expressing
mutant H-RAS V12 (C10). Total β-actin protein expression was blotted in the
same membrane as a loading control. Total protein levels of ERK1/2, AKT
and JNK1/2 did not differ between the cell lines (data not shown). Data are
from a representative experiment (n = 3–5).
Figure 3 (Right). Phosphorylation of P-JNK1/2, P-ERK1/2, and
P-AKT S473 in HCT116 cells after a 1 Gy radiation exposure. (A)
JNK1/2 and p38 phosphorylation in WT HCT116 cells after a 1 Gy
exposure. Cells were serum-starved for 24 h, irradiated (1 Gy)
and samples taken at different time points (0–24 h) and homoge-
nized for SDS PAGE and immunoblotting as indicated in Materials
and Methods. A representative experiment (n = 3) is shown. (B)
Alteration of ERK1/2 phosphorylation 0–24 h after a 1 Gy exposure
in HCT116 cells (WT), mutant K-RAS-deleted cells (C2) or mutant
K-RAS-deleted cells expressing mutant H-RAS V12 (C10). Data are
a representative from three experiments. (C) Alteration of AKT
S473 phosphorylation 0–24 h after a 1 Gy exposure in WT, C2
and C10 cells. Data are a representative from four experiments.
A
B
C
A
B
C

Page 5

percentage of cells present in G1phase but prevented radiation-induced
alterations in G1and S phase cell numbers (Fig. 4B). Inhibition of PI3K
function increased the percentage of cells present in G1phase and reduced
cell numbers in S phase, which were, again, not altered following radiation
exposure (Fig. 4B). Collectively, these findings argue that in HCT116 cells
inhibition of radiation-induced signaling by either ERK1/2, AKT or
JNK1/2 blocks the stimulation of G1progression into S phase by low doses
of radiation.
Radiation-induced ERK1/2, JNK1/2 and PI3K signaling regulates the
expression of cyclin molecules in parental HCT116 cells. Signaling by the
ERK1/2, PI3K/AKT and JNK1/2 pathways has been linked by many labo-
ratories to enhanced expression of cyclin molecules; particularly those
required for G1progression and entry into S phase. Based on our findings
in Figures 3 and 4, we examined whether ERK1/2, JNK1/2 and PI3K/AKT
signaling was responsible for the altered expression of cyclin D, cyclin E and
cyclin A in HCT116 cells.
Basal levels of cyclin expression were different in cells expressing K-RAS
D13, H-RAS V12, or without activated RAS protein expression (Fig. 5A).
Loss of K-RAS D13 expression reduced basal expression of cyclin A but
enhanced the levels of cyclin D1 and cyclin E. Expression of H-RAS V12
did not restore cyclin A expression, partially blunted elevated cyclin D1
levels and further stimulated expression of cyclin E.
Further studies then examine the impact of radiation on cyclin levels
over a 24h time course. Radiation stimulated the expression of cyclin D and
cyclin A in parental HCT116 cells, but not in mutant K-RAS deleted cells
or H-RAS V12 cells (Fig. 5B and C). Parental HCT116 cells expressed high
basal levels of cyclin E, which was modestly enhanced 24 h after exposure in
all HCT116 cell lines (Fig. 5D). Inhibition of MEK1/2 over 24 h gradually
reduced basal expression of cyclin A, cyclin D and cyclin E and abolished
radiation-stimulated expression of cyclin A, cyclin D and cyclin E (Fig. 5E).
Inhibition of JNK1/2 gradually reduced basal expression of cyclin A and
abolished radiation-stimulated expression of cyclin A without altering expres-
sion of other cyclin proteins (Fig. 5F). Inhibition of PI3K reduced basal
expression of cyclin D, and abolished radiation-stimulated expression of
cyclin D (Fig. 5F). Collectively, these findings demonstrate that coordinated
signaling by ERK1/2, JNK1/2 and PI3K contributes to radiation-induced
cyclin expression and S phase entry in parental HCT116 cells.
K-RAS and H-RAS signaling differentially modulate the radiation-
induced expression of p21, p53 and hMDM2 in HCT116 cells. Ionizing
radiation at modest to high doses (>2 Gy) has been shown to cause a
p53-dependent growth arrest in cells which has been linked to enhanced
expression of the cyclin kinase inhibitor protein p21. Based on our findings
in Figures 5 and 6, additional studies examined the expression of p21, p53,
p53 S15 phosphorylation and hMDM2 in HCT116 cell lines. Radiation
(1 Gy) increased the expression of p21, p53 and hMDM2, and increased
p53 Serine 15 phosphorylation, in parental HCT116 cells (Figs. 6A–D). Of
note, with relevance to the cyclin expression studies presented in Figure 6,
expression of p21 was maximal 2–3 h after exposure and had declined to
much lower levels within 6–9 h.
To our surprise, deletion of mutant K-RAS D13 largely abolished radia-
tion-stimulated expression of p21, p53 and hMDM2 without altering the
stimulation of p53 S15 phosphorylation by radiation. In H-RAS V12 cells,
Radiation, RAS and Cell Cycle Regulation
460 Cell Cycle 2005; Vol. 4 Issue 3
Figure 4. Radiation (1 Gy) promotes modest S phase entry in growth arrest-
ed parental HCT116 cells, but not in mutant K-RAS deleted (C2) or H-RAS
V12 (C10) cells, that is blocked by inhibitors of MEK1/2, PI3K and
JNK1/2. Cells were serum-starved for 24 h, where indicated then treated
with either vehicle (DMSO), PD184352 (2 µM), SP600125 (1 µM) or
LY294002 (1 µM) and 30 min later irradiated (1 Gy) or mock exposed.
Cells were isolated 24 h after irradiation, fixed, digested with RNAse,
stained with propidium iodide and subjected to flow cytometery to deter-
mine the percentage of cells in each phase of the cell cycle, as described
in Methods. (A) Cell cycle profiles of unirradiated and irradiated WT, C2
and C10 cells, 24 h after exposure. Bars represent mean ± SEM from four
experiments (*p<0.05 less than corresponding unirradiated cell value;
#p<0.05 greater than corresponding unirradiated cell value). (B) Inhibition
of MEK1/2, PI3K or JNK1/2 prevents the radiation-induced reduction in cell
numbers in G1phase and the radiation-induced increase in cell numbers in S
phase, 24 h after exposure. Bars represent mean ± SEM from four experi-
ments (*p<0.05 less than corresponding unirradiated cell value; #p<0.05
greater than corresponding unirradiated cell value).
Figure 5 (Right). Radiation-induced expression of cyclin D1 and cyclin A is
dependent on enhanced MEK1/2, PI3K and JNK1/2 signaling in parental
HCT116 cells. Cells were cultured and exposed to radiation as described in
the legend to Figure 5. Cells (WT, C2, C10) were isolated and lysed for
Western blotting using specific antibodies for: (A) Total cyclin expression at
t = 0 in unirradiated cells: Bars represent mean ± SEM from four experiments
(*p<0.05 less than corresponding parental (WT) cell value; #p<0.05
greater than corresponding parental (WT) cell value); (B) Cyclin A levels
0–24 h after exposure, (C). cyclin D1 levels 0–24 h after exposure and (D)
cyclin E levels 0–24 h after exposure. β-actin was blotted in the same
membrane as a loading control. Densitometry values were normalized respect
to β-actin and expressed as percentage of the cyclin expression at t = 0 in
the respective mock exposed cells. Error bars represent mean ± SEM from
three or four experiments. (E) Impact of MEK1/2 inhibition on the increase
in the expression of cyclins induced by 1 Gy in HCT116 cells. HCT116 cells
were serum-starved for 24 h, treated with PD184352 (2 µM) or vehicle
(DMSO) 30 min prior to irradiation. Homogenates were obtained 12 or 24 h
after radiation and the samples were subjected to SDS PAGE to determine
expression of cyclins A, D1 and E. The Figure shows a representative exper-
iment (n = 3). (F) Effect of JNK1/2 inhibition or inhibition of PI3K (AKT
activation) using the JNK1/2 inhibitor SP600125 (1 µM) and the PI3K
inhibitor LY294002 (1 µM), respectively, on the increase in the expression
of cyclins induced by 1 Gy in HCT116 cells. HCT116 cells were serum-
starved for 24 h, and inhibitor drugs added 30 min prior to irradiation.
Homogenates were obtained 12 or 24 h after radiation and the samples
were subjected to SDS PAGE to determine expression of cyclins A and D1.
The Figure shows a representative experiment (n = 3).
A
B