From the Department of Experimental Pathology and
Oncology, University of Firenze, Viale G. B. Morgagni 50, 50134 Firenze, Italy, the
Department of Biological Science,
University of Tulsa, Tulsa, Oklahoma 74104-3189, the
§ Department of Clinical Physiopathology, University of
Firenze, Viale Pieraccini 6, 50134 Firenze, and the ¶ Department
of Biotechnology and Biosciences, University of Milano Bicocca, Piazza
della Scienza 2, 20126 Milano, Italy
Received for publication, October 22, 2002, and in revised form, November 12, 2002
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ABSTRACT |
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The role of K+ channel activity
during cell cycle progression has become a research topic of
considerable interest. Blocking of K+ channels inhibits the
proliferation of many cell types, although the mechanism of this
inhibition is unclear. There is speculation that K+
channels differentially regulate the electrical potential of the plasma
membrane (Vm) during proliferation. We have demonstrated that in tumor cells the value of Vm is
clamped to rather depolarized values by K+ channels
belonging to the HERG family. We report here that tumor cell lines
preferentially express the herg1 gene and a truncated, N-deleted form that corresponds to herg1b. This
alternative transcript is also expressed in human primary acute myeloid
leukemias. Both HERG1 and HERG1B proteins are expressed on the plasma
membrane of tumor cells and can form heterotetramers. The expression of HERG protein isoforms is strongly cell cycle-dependent,
accounting for variations in HERG currents along the mitotic cycle.
Moreover, the blocking of HERG channels dramatically impairs cell
growth of HERG-bearing tumor cells. These results suggest that
modulated expression of different K+ channels is the
molecular basis of a novel mechanism regulating neoplastic cell proliferation.
Potassium channels are the most diverse class of plasma membrane
ion channels, and this hetereogeneity is reflected by the large variety
of specific roles they exert in different cell types. Besides the
regulation of excitability in nerve and muscle cells, and the linkage
between plasma membrane and metabolic activity, there is now evidence
that K+ channels are involved in the regulation of cell
proliferation (1). The cellular mechanisms linking K+
channel activity and cell proliferation remain unclear, although a
possibility is that activation of K+ channels might be
required for the passage of cells through a specific phase of the
mitotic cycle (1, 2). K+ channel blockage has been shown to
be antiproliferative for numerous non-excitable as well as excitable
cells (3-9); however, the link between K+ channel activity
and cell cycle progression remains elusive. One hypothesis is that
K+ channels might regulate cell volume, as well as the
concentration of intracellular solutes critical for cell metabolism;
alternatively, K+ channel activity might serve to maintain
permissive membrane potentials at critical cell cycle checkpoints (1).
Furthermore, terminally differentiated G0 cells display a
hyperpolarized value of their membrane potential
(Vm), whereas cycling and in particular tumor cells
are quite depolarized (10).
We have shown previously (11) that the depolarized state of many tumor
cell lines can be explained by the lack of classical inward rectifier
K+ channel-type inward rectifier K+ currents
accompanied by the expression of peculiar voltage-dependent K+ channels, belonging to the
HERG1 family (12, 13). The
herg (human eag-related) gene belongs to an
evolutionarily conserved multigenic family of voltage-activated K+ channels, the eag (ether
a-gò-gò) family (15). herg genes and HERG
currents (IHERG) are preferentially expressed in neoplastic cell lines of different histogenesis, as well as in primary human endometrial cancers (11, 14). The functional properties of HERG
channels are complex, and their contribution to the repolarization of
the cardiac action potential well understood (16). For our purposes,
however, it is sufficient to recall that the HERG activation and
inactivation curves are such that their crossover produces maximal
channel open probability between The molecular basis of IHERG is being uncovered. HERG
channels are tetramers, with each subunit consisting of six
transmembrane domains, and both N and C termini are located
intracellularly. The HERG proteins compose the We thus investigated the molecular structure of herg genes
and HERG proteins in tumor cell lines. In particular, because
IHERG biophysical features (rapid deactivation kinetics and
strong dependence of the activation gate on depolarized values of the
Vm) as well as herg biomolecular
characteristics (presence of multiple RNA bands ranging from 4.4 to 1.9 kDa as revealed in Northern blot experiments) in tumor cells are quite
different from those displayed by the channel in the heart and in
herg1-transfected cells (11, 30), the expression of
different herg genes, as well as of alternate transcripts in
tumor cells, was investigated.
We report here that tumor cell lines, as well as primary human tumors,
preferentially express the herg1 gene, along with
herg1b. Both the full-length HERG1 and HERG1B proteins are
coexpressed and can form heterotetramers on the plasma membrane of
tumor cells. The expression of the two HERG protein isoforms turned out
to be strongly cell cycle-dependent, suggesting a possible
explanation for the variations in IHERG along the mitotic
cycle previously demonstrated in neuroblastoma cells (12). Moreover,
the block of HERG channels dramatically impaired cell growth of
HERG-bearing neuroblastoma cells.
On the whole, these results contribute to an understanding of the
molecular basis of a novel mechanism regulating neoplastic cell
proliferation, i.e. HERG K+ channels.
Cell Culture--
The human neuroblastoma SH-SY5Y and LAN1 clone
AE12 (kindly provided by Dr. G. Mugnai, University of Firenze, Italy)
cell lines, the human rhabdomyosarcoma RD12 cell line (kindly provided by Dr. P. L. Lollini, University of Bologna, Italy), and HEK 293 cells (kindly provided by Dr. S. Heinemann, University of Jena, Germany) were cultured in DMEM containing 4.5 g/liter of glucose and
10% FCS (HyClone) and incubated at 37 °C in a humidified atmosphere with 5% CO2. The human colon carcinoma H630 (kindly
provided by Dr. E. Mini, University of Firenze, Italy), the human
monoblastic leukemia FLG 29.1 (kindly provided by Dr. P. A. Bernabei, Hematology Unit, Firenze, Italy), and the human mammary
adenocarcinoma SkBr3 and the human retinoblastoma Y-79 (kindly provided
by Dr. A. Albini, IST, Genova, Italy) cell lines were all cultured in
RPMI 1640 medium containing 5, 10, and 20% FCS, respectively, and
incubated at 37 °C in a humidified atmosphere with 5%
CO2.
Cell Transfection--
The HEK 293 cells, cultured on 100-mm
Petri dishes, were transiently transfected with herg1
cDNA cloned into HindIII/BamHI sites of the
pCDNA3.1 vector (Invitrogen) by the calcium phosphate method. Six
hours before transfection the medium was replaced once. The
precipitation solution was then added to the cell cultures. The
precipitation solution was 400 µl of 2× BES-buffered saline (50 mM BES, 280 mM NaCl, 1.5 mM
Na2HPO4·2H2O (pH 6.96)) plus 400 µl of 0.25 M CaCl2 and 36 µg of the
cDNA construct. The medium was replaced 15 h later. Protein
extraction, as well as control patch clamp analysis, was performed
48-72 h post-transfection. pCDNA3.1 without the insert was also
transiently transfected as above in the same cell line, which was used
as a negative control. These cells are referred to as MOCK.
RNase Protection Assay--
The RNase Protection Assay (RPA) was
performed essentially according to Dixon and McKinnon (31). Briefly,
RNA was extracted from semiconfluent tumor cell lines (see above) by
the guanidinium/isothiocyanate method (32). Commercially available
human brain RNA (Clontech) as well as human heart
RNA (Ambion) were used as controls for herg1 and
herg3 expression and for herg1b, respectively.
Thirty µg of total RNA was hybridized overnight at 48 °C with
[32P]UTP-labeled RNA probes. Digestion was then performed
for 1 h at room temperature with RNase A (40 µg/ml) and T1 (2 µg/ml). Yeast tRNA (Invitrogen) was used as a negative control to
test for the presence of probe self-protection bands. The samples were run on a 6.6% polyacrylamide gel and exposed for 1-8 days. The human
erg probes used were the following: herg1
(nucleotides 1401-1880, GenBankTM accession number
NM 000238); herg2 (nucleotides 2041-2245,
GenBankTM accession number NM 030779); herg3
(nucleotides 272-480, GenBankTM accession number AF
032897); the N-terminal herg1 clone (nucleotides 184-589,
GenBankTM accession number NM 000238) named
hergN135 was produced in our laboratory (14).
The probe relative to herg1b was produced by RT-PCR (see
below) from the FLG 29.1 cell line. Human cyclophilin (Ambion) was used
as an internal loading control.
Reverse Transcription-PCR--
Two µg of total RNA was
retrotranscribed with Superscript reverse transcriptase (200 units)
(Invitrogen) in the presence of random hexamers (2.5 µM).
For herg1b amplification, the cDNA thus obtained was
amplified using HotStarTaq polymerase (5 units) (Qiagen) and the
following primers: herg1b-up 5'-CGATTCCAGCCGGGAAGGC-3'; herg1b-down
5'-TGATGTCCACGATGAGGTCC-3' (product size, 363 bp), according to the
sequence reported in Lees Miller et al. (28). It is worth
noting that herg1b-up maps on exon 1b of the genomic sequence, whereas
herg1b-down maps on exon 6 of the same sequence that is shared by both
herg1b and herg1 isoforms. Thirty five cycles of
amplification were carried out after 15 min of enzyme activation at
95 °C as follows: 94 °C for 1 min, 56 °C for 1 min, 72 °C
for 1 min. The PCR product was cloned by means of the TA cloning system
(Invitrogen) and then subcloned in pBluescript vector, sequenced, and
used for RPA experiments as reported above. PCRs relative to human
eag and Kv 1.3 transcripts were performed according to Smith et al. (33), using the following primers: heag-up 5'-CGCATGAACTACCTGAAGACG-3'; heag-down
5'-TCTGTGGATGGGGCGATGTTC-3' (product size, 479 bp); Kv 1.3-up
5'-TCGAGACGCAGCTGAAGAC-3'; Kv 1.3-down 5'-GGTACTCGAAGAGCAGCCAC-3'
(product size, ~350 bp).
Cloning of Herg1b from Tumor Cells--
The hergb
transcript was cloned from FLG 29.1 cells with two different
approaches, RT-PCR and RACE-PCR. Briefly, poly(A) RNA was extracted by
means of a poly(A) Pure kit (Ambion), and cDNA-retrotranscribed using either oligo(dT) primers (for RT-PCR cloning) or the 3'-adapter primer according to the 3'-RLM-RACE kit protocol (Ambion) (for RACE-PCR). PCR was performed with Takara polymerase (2.5 units) using
the following primers: UP primer, 5'-AGGGAGCCAAGTCCTCCATGG-3' (which
maps into the sequence relative to the human exon 1b (dbEST Id:
8445856)); DOWN primer, 5'-GCGGCCGCACTGCCCGGGTCCGAG-3' (which maps at
the end of the herg1 sequence with the addition of a
NotI restriction site). The amplified band of ~2.5 kb was
purified and cloned into the pCR2.1 vector (Invitrogen) using the TA
cloning kit (Invitrogen). 3'-RACE PCR was performed according to the
protocol provided in the Ambion kit, using 2.5 units of HotStarTaq
polymerase (Qiagen), and using the same UP primer as reported above for
first round. The second round of amplification was performed using
herg1b-specific primers: UP primer
5'-CAGGCAAAGCTTAGGGAGCCAAGTCCTCCATGG-3' (which corresponds to
the above reported up primer used for RT-PCR plus a HindIII
restriction site); DOWN primer 5'-CAGCGCGCGGCCGCCTGGGTGAGCCACGTGTC-3' (which maps in the untranslated region of the herg1 sequence
(see above) and contains a NotI restriction site). In this
case the amplified band was cloned into
HindIII/NotI sites of pBluescript (Stratagene)
vector. All the cloned bands were sent off for sequencing by PRIMM DNA
Sequencing Service.
Protein Chemistry--
For Western blot experiments both total
cell lysate and membrane proteins were used as described previously
(34). For membrane decoration two anti-HERG antibodies were used: an
anti-ERG antibody raised against the C terminus (residues 1121-1137 of
rat ERG1, Alomone Labs) and an anti-HERG antibody against the N
terminus (residues 1-135 of human ERG1) developed in our laboratory
(14). The latter serum was immunopurified on a column preadsorbed with the antigen and tested by means of enzyme-linked immunosorbent assay.
N-Glycosidase F (Roche Molecular Biochemicals) was used following the manufacturer's instructions. For proteinase K (Roche Molecular Biochemicals) treatment, confluent cell cultures, seeded on
100-mm Petri dishes or on 25-cm2 flasks, were washed with
PBS and incubated with 3 ml of a solution containing 10 mM
HEPES, 150 mM NaCl, and 2 mM CaCl2
(pH 7.4) with or without 200 µg/ml proteinase K, at 37 °C for 30 min; enzyme activity was then stopped with 2 ml of ice-cold PBS
containing 6 mM phenylmethylsulfonyl fluoride, 25 mM EDTA. After three washes in ice-cold PBS, membrane
proteins were isolated and processed as above. For immunoprecipitation
3 mg of total protein lysate was cleared by incubation with protein
A-Sepharose (50 µl of a 50% slurry) for 2 h at 4 °C.
Anti-HERG antibody against the N terminus was added, and the samples
were incubated on ice for 1 h. 40 µl of protein A-Sepharose was
then added, and each sample was incubated overnight at 4 °C. The
immunoprecipitates were washed in lysis buffer and ice-cold PBS prior
to SDS-PAGE. Membranes were then immunoblotted with anti-ERG C terminus
antibody. Super Signal (Pierce) was used for blot visualization.
Cell Synchronization and Cell Cycle Analysis--
SH-SY5Y
neuroblastoma cells were synchronized by hydroxyurea treatment
according to Arcangeli et al. (12). Retinoic acid treatment
was performed according to Arcangeli et al. (35). The
distribution in the cell cycle phases was determined by flow cytometry;
samples of cell suspension (106 cells/ml) were stained with
propidium iodide (PI) as described by Vindeløv and Christensen (36).
The samples were then analyzed using a FACScan flow cytometer (BD
Biosciences) equipped with a 5-watt argon ion laser. The fluorescence
of PI-stained nuclei was excited at 488 nm, and histograms of the
number of cells versus linear integrated red fluorescence
were recorded for 50,000 nuclei/sample. DNA histograms were analyzed
using the MultiCycle DNA content and cell cycle analysis software
(Phoenix Flow Systems, San Diego).
Cell Proliferation Assay--
The human neuroblastoma cell lines
SH-SY5Y and LAN1, cultured as above, were seeded in 96-well plates
(Corning Glass) at a cell density of 1.8 × 104 and
1.2 × 104 cells per well, respectively, and then
starved for 16 h in DMEM containing 1% FCS. After this time, DMEM
containing 2.5% FCS, with or without HERG channel blocker (E4031 200 or 50 µM, and WAY 123,398, 50 µM, final
concentrations), was added, and this was considered to be the time 0 of
the experiment. At different times of incubation, cells were assayed
using the colorimetric Cell Proliferation Reagent WST-1 (Roche
Molecular Biochemicals), whose tetrazolium salt is cleaved by
mitochondrial enzymes so that the amount of dye developed (read at 450 nm, reference at 630 nm) directly correlates to the number of
metabolically active cells in the culture. Absorbance of culture medium
plus wst-1 in the absence of cells was the blank position for the
enzyme-linked immunosorbent assay reader (ELx-800, Biotek Instruments).
Data Acquisition and Analysis--
RPA and Western blot images
were acquired by an HP4C scanner, and the relative bands were analyzed
by Scion Image software.
Experiments performed were aimed at determining the molecular
basis of HERG currents in tumor cells. The first point to be explored
was whether cancer cells expressed different herg genes, namely herg1, herg2, or herg3. For
this purpose, RPA experiments were performed using appropriately cloned
herg1, -2, and -3 probes on tumor cell
lines of different histogenesis: human neuroblastoma (SH-SY5Y), human
rhabdomyosarcoma (RD12), human colon carcinoma (H630), human mammary
carcinoma (SkBr3), and human monoblastic leukemia (FLG 29.1). The
results of these experiments are shown in Fig.
1. As shown in Fig. 1A, all of
the tumor cell lines tested express the herg1 gene, although
at different intensities (see also the densitometric analysis reported
in Fig. 3a). In particular, both SH-SY5Y and FLG 29.1 cells appear to overexpress the herg1 gene, as suggested
previously (11). On the other hand, no human tumor cell line expresses
the herg2 gene (Fig. 1B), except for the
human retinoblastoma cell line Y-79. This expression, which represents
the positive control in our experiments, is in keeping with the well
known expression of erg2 gene in the retina, at least in rat
(20). As for herg3 expression (Fig. 1C),
only SkBr3 cells express the gene at good levels, as compared with
human brain. Therefore, these cells express both herg3 and
herg1, with the latter expressed at relatively low
intensity.
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30 and
50 mV in resting conditions
(12, 13), thus contributing substantially to the resting potential of
tumor cells (12, 13). In some neurons, the HERG role appears to be the
regulation of the action potential firing frequency (17). Recent
studies (18) indicate that in various normal tissues other than heart
and brain, IHERG and the erg gene are expressed
only at very early stages of embryo development and are subsequently
replaced by inward rectifier K+ channel currents.
subunit of the
channel, whereas a
subunit associating with HERG is represented, at
least in parts of the heart, by the MIRP1 protein (19). Three different ERG proteins have been cloned in mammals: ERG1, ERG2, and ERG3 (HERG1,
HERG2, and HERG3 in humans), with the latter being specific to the
nervous tissues (20). The recently characterized genomic structure of
the herg gene encoding the HERG1 protein (herg1
gene) consists of 15 exons, spanning about 19 kb on chromosome 7 (21, 22). Most of the exons code for the N and C termini, which therefore appear to be putative sites for alternative splicing. The HERG1 C
terminus contains the cyclic nucleotide binding domain (15), and an
alternatively spliced product of this region (named
HERGUSO) has been identified in the heart (23), which
cannot be expressed on the plasma membrane by itself but could modify
the biophysical properties of IHERG. Conversely, the N
terminus is made up of two domains, the "eag" domain, comprising
the first 135 amino acids of the HERG1 sequence, and the "proximal"
domain, which extends from position 135 to about position 366. The
former domain, a eukaryotic PAS domain, is involved in the regulation
of channel gating (24-26), particularly with regard to deactivation
rates, whereas the latter is apparently involved in regulating channel activation. An alternative transcript of the herg1 gene,
displaying a short N terminus, has been identified in mouse and human
hearts, merg1b and herg1b, respectively (27, 28).
Compared with merg1, merg1b has a different first
exon (designated 1b) located between exons 5 and 6 of the
Merg1 genomic sequence. Because the region upstream from
exon 1b may contain an alternate transcription initiation site, it is possible that merg1b represents an alternate
transcript more than a splicing variant (27, 28). However, recent
evidence (29) seems to exclude the expression of this transcript at the protein level in the hearts of various species.
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Fig. 1.
herg-1, -2, and
-3 expression in various human tumor cell lines.
RNA extracted from SH-SY5Y human neuroblastoma cells, RD12 human
rhabdomyosarcoma cells, H630 human colon carcinoma cells, SKBr3 human
mammary carcinoma cells, and FLG 29.1 human monoblastic leukemia cells
was probed with the herg1, -2, and -3 probes as described under "Materials and Methods." Human brain RNA
was used as a control for herg1 and herg3,
whereas RNA from human retinoblastoma Y-79 cells was used as a control
for herg2 expression. Human cyclophilin (hcyc)
(Ambion) was used as an internal control and yeast tRNA as a negative
control to test for probe self-protection bands. A,
herg1 (1-day exposure); B, herg2
(5-day exposure); C, herg3 (1-day exposure). The
protected bands corresponding to the above-mentioned genes are
indicated by an arrow.
Because Kv channel encoding genes other than herg, like
eag or Kv 1.3, have reportedly been linked to
cell proliferation in different models (37, 38), we tested whether the
above-mentioned genes were overexpressed in the tumor cell lines under
study. Fig. 2 shows the expression of
eag and Kv 1.3 genes in SH-SY5Y, FLG 29.1, H630,
RD12, and SkBr3 cells, as detected by RT-PCR. It is evident that,
despite the good quality of all the cDNAs tested (see
gapdh expression in the lower panel of Fig. 2),
only SH-SY5Y, as reported previously (39), and RD12 cells, as expected (40), express the eag gene. Kv 1.3 is not
expressed in any of the tumor cell lines examined, although it is
present in human peripheral resting lymphocytes as reported previously
(33).
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On the whole, data presented in Figs. 1 and 2 demonstrate that cancer cell lines preferentially express the herg1 gene. Neither herg2 nor -3 nor the other Kv encoding genes that have been proposed to play a role in the control of cell proliferation (i.e. eag and Kv 1.3) are expressed at the RNA level, irrespective of the histological origin of the cancer cell lines tested. This result rules out the possibility that the herg RNA profile (11) as well as the HERG biophysical features specific of the different tumor cell lines that we have tested are due to coexpression of different proportions of the products of the three herg genes. The possibility of deletions as well as alternative splicing products of the herg1 gene in tumor cells was then investigated. Because tumor IHERG was demonstrated previously (30) to display fast deactivation kinetics, a feature associated with a deletion in the N-terminal domain, we first looked for the existence of herg1 deletions and/or splicing modifications at this level.
A probe was constructed (N(135) herg1 terminus)
for RPA experiments, comprising the first 135 amino acids of the HERG1
sequence, i.e. the eag domain. The results of
this experiment are reported in Fig. 3.
The eag domain is present in the herg1 transcript
of all the tumor cell lines tested; however, when comparing the
densitometric analysis of the results obtained with the
herg1 probe, encompassing a conserved region of the gene
(Fig. 1), with the densitometric analysis of the experiments performed
with the N(135) herg1 terminus probe (see Fig.
3, a and b), it is evident that the
eag domain is expressed at a lower level, especially in
SH-SY5Y and FLG 29.1 cells. A possible explanation of these data is
that tumor cells express both a full-length herg1 mRNA,
and a truncated form of the latter, lacking part or the entire N
terminus.
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The possibility that such N-truncated RNA could belong to
the already identified herg1 alternative transcript named
herg1b was then tested. First, the expression of
herg1b was studied in two different tumor cell lines
(SH-SY5Y and FLG 29.1 cells) by RT-PCR. As shown in Fig.
4A, herg1b mRNA
is indeed expressed in both the cell lines tested. The possibility of
the simultaneous expression of herg1 and herg1b
in tumor cell lines was then investigated by constructing probes for
RPA experiments comprising first the entire herg1b exon and
part of exon 6, which is shared by herg1 and
herg1b genes (27, 28). If both herg1 and
herg1b are expressed in tumor cells, two RPA bands would be
expected with molecular weights 258 and 363 bp, respectively. This
result indeed occurred (see Fig. 4B) both in SH-SY5Y and FLG
29.1 cells and in the heart. Note that this is not common to all
tissues expressing herg1, as only a lower RPA band was
detectable in brain RNA, corresponding to the herg1 gene.
Moreover, observing the two bands present in FLG 29.1 cells, it is
evident that the upper band (attributable to herg1b) has a
higher intensity compared with the lower band corresponding to
herg1. As tumor cell lines are deregulated in terms of their
RNA expression, we analyzed whether herg1b mRNA could be
detected in primary human tumors. We demonstrated recently (41)
that the herg1 gene is expressed in human myeloid leukemias; hence, we chose these cells as samples because they are not
contaminated by other cell types, such as stromal or smooth muscle
cells, that could express the herg1b transcript (27, 28). As
shown in Fig. 4C, all of the primary myeloid leukemias we
tested expressed the herg1b exon, ruling out the possibility
that such expression is exclusively an artifact related to the altered
gene expression occurring in established tumor cell lines.
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The nature of the transcript containing the herg1b exon in tumor cells was then investigated by cloning the entire transcript from tumor cells. Clones obtained by RT-PCR and 3'-RACE PCR (see "Materials and Methods") were sequenced demonstrating that tumors cells do express the entire herg1b alternative transcript (GenBankTM accession number AJ512214). The herg1b transcript cloned from tumor cells was identical to that identified in human heart (27, 28), except for two polymorphisms (in position 689 and 953 of the submitted sequence), and identical to that reported for the herg1 sequence cloned from neuroblastoma cells (11). It is worth noting here that, as stated in the Introduction and reported under "Materials and Methods," the sequence of the herg1b exon was confirmed on the genomic sequence of chromosome 7, suggesting the possibility that herg1b represents an alternate transcript more than a splice variant.
Furthermore, because data gathered from our RPA experiments showed that, in FLG 29.1 cells, herg1b represented the greatest amount of the total HERG mRNA, the next step was to determine whether the encoded protein HERG1B was expressed on the plasma membrane. Western blot experiments were, therefore, performed on SH-SY5Y neuroblastoma and FLG 29.1 leukemia cells, using anti-HERG antibodies, specific for both the C and N termini (see "Materials and Methods").
When experiments were performed with an anti-C terminus antibody (Fig.
5A), two main bands were
detectable in herg1-transfected cells, weighing 135 and
about 155 kDa, respectively, as expected (29, 42). On the other hand,
in SH-SY5Y and FLG 29.1 cells, two main groups of bands could be seen:
an upper group ranging from 135 to ~155 kDa, and a lower group
ranging from 85 to ~100 kDa. It is worth noting that the upper group
of bands is more evident in SH-SY5Y cells, whereas they are barely
detectable in FLG 29.1 cells. The possibility that both the two groups
represented HERG proteins expressed on the plasma membrane was then
tested by performing experiments on cells treated with specific enzymes used to evaluate the glycosylation state (N-glycosidase F)
as well as the plasma membrane expression (proteinase K) of HERG proteins. As shown in Fig. 5B, when membrane extracts from
both SH-SY5Y and FLG 29.1 cells were treated with
N-glycosidase F (lanes 1), both the bands of
~155 and those ~100 kDa shifted to lower molecular weights.
Furthermore, when cells were treated with proteinase K (Fig.
5B, lanes 3) both the bands of ~155 and those
of ~100 kDa disappeared, and only the bands of ~135 and ~85 kDa
could be seen. The results were similar in both cell lines tested; the only difference was that all the bands of lower molecular weight were
preferentially expressed in FLG 29.1 cells, whereas those of higher
molecular weight were observed only after longer exposure of the
autoradiographic film (see Fig. 5B, inset).
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The results of these experiments suggest that both SH-SY5Y and FLG 29.1 cells express two HERG isoforms on their plasma membranes: one corresponding to the full-length protein (135 kDa), and its various glycosylated forms (the bands of ~155 kDa); and the second corresponding to HERG1B, which has a molecular mass of 85 kDa in the unglycosylated, immature form, and various glycosylated forms expressed on the plasma membrane (the bands of ~100 kDa). To verify this, experiments were performed using the antibody directed against the HERG N-terminal domain (see "Materials and Methods"). As shown in Fig. 5C, the reactivity of this antibody on HERG-transfected HEK 293 cells is comparable with that of the anti-C-terminal antibody (compare C with A), whereas in SH-SY5Y cells only the bands of higher molecular mass, ranging from 135 to 155 kDa, can be detected with this antibody. Similar results were obtained in FLG 29.1 cells with very long exposure (not shown).
On the whole, it appears plausible to conclude that tumor cell lines
express two mature, highly glycosylated HERG proteins on their plasma
membrane, a full-length HERG1 and the HERG1B isoform. In order to
explore the possibility that these two proteins could form
heterotetramers in cancer cells, as observed in transfected oocytes
(27, 28), immunoprecipitation experiments were performed. Proteins from
both herg1-transfected HEK 293 cells and SH-SY5Y cells were
immunoprecipitated using the anti-N-terminal antibody that recognizes
only the herg1 product, and the subsequent Western blot was
decorated with the anti-C-terminal antibody that is capable of
recognizing both isoforms. As shown in Fig.
6, the immunoprecipitated proteins
contain only HERG1 full-length in the transfected HEK 293 cells
(lane 2), whereas bands indicating the presence of both HERG1 and HERG1B proteins are detected in SH-SY5Y cells (lane 4).
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It is, therefore, likely that tumor cell lines not only express both HERG1 and HERG1B proteins on their plasma membrane but that these proteins can form heterotetramers in these cells. Such heterotetramers can be composed of different amounts of each single protein, as suggested by the different appearance of Western blots on SH-SY5Y cells and FLG 29.1 cells.
We have demonstrated previously (12) that biophysical features, such as
the activation voltage, of the HERG currents in neuroblastoma cells are
cell cycle-dependent. The possibility now exists that such
features can be accounted for by differential expression of HERG1B and
full-length HERG1 proteins. Therefore, SH-SY5Y cells were synchronized
by treatment with hydroxyurea (HU) and retinoic acid (RA), as reported
previously (12). After a 15-h treatment with HU, neuroblastoma cells
were blocked at the G1/S boundary. Then, after HU
withdrawal, cells enter almost synchronously into S phase, so that
after 6 h a high percentage of cells is in the middle of the S
phase and reaches the S/G2 boundary after 8 h (Fig.
7A). Western blot experiments
performed on the same cell preparation (Fig. 7B) showed that
expression of the HERG1B mature form is significantly up-regulated in
the middle of S phase as compared with unsynchronized cells, and to G1/S HU-blocked cells (see also the densitometric analysis
of the two HERG isoforms reported in inset b). Conversely,
when SH-SY5Y cells were treated for 11 days with RA, a strong
synchronization of the cells in G1 was achieved (Fig.
8A) (12). Under these conditions, a strong up-regulation of HERG proteins can be detected in
Western blot experiments (Fig. 8B) in agreement with results reported previously (35). This up-regulation apparently involves both
of the isoforms, although a stronger increase in the intensity of the
mature full-length HERG1 band can be observed (see the densitometric
analysis reported in inset b).
|
|
On the whole, results presented in Figs. 7 and 8 are consistent with the previously reported modulation of IHERG activation curves during the cell cycle (12); in fact, prevalence of HERG1B expression shifts the activation voltage toward depolarized values (see the mouse erg1b encoded currents reported in Refs. 27 and 28), whereas the IHERG of the full-length HERG has an activation voltage that is more hyperpolarized.
These results allow us to ask a fundamental question: what is the
putative role of IHERG in the regulation of cancer cell proliferation? A preliminary answer to this question was obtained by
testing the effects of a specific IHERG inhibitor (E4031)
on proliferation of neuroblastoma cells, by utilizing the same approach used previously (41) in acute myeloid cells. The results of these
experiments are reported in Fig. 9; it is
evident that E4031 significantly impairs proliferation in SH-SY5Y cells
(left panel), whereas it does not significantly affect cell
growth in the LAN1 clone AE12 cells (right panel), which do
not express
IHERG.2 Similar
results were obtained with another HERG blocker, namely Way 123,398 (see insets in Fig. 9), in a different set of experiments. IHERG clearly affects proliferation of the SH-SY5Y cells;
however, because the currents of both HERG1 and HERG1B proteins are
blocked by these compounds, no conclusion about the differential role of the two HERG isoforms in cell growth can be inferred.
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DISCUSSION |
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Results reported in this paper clearly show that various tumor cell lines preferentially express the herg1 gene along with a truncated form of the HERG1 protein that corresponds to the alternative transcript of herg1 first discovered in the heart, herg1b. The herg1b isoform is also expressed in primary human tumors. Moreover, the data reported here show that the corresponding proteins, HERG1 and HERG1B, are differentially expressed during cell cycle phases and that HERG currents are capable of modulating cell proliferation in tumor cells.
The expression of herg1, -2, and -3 was studied in various human tumor cell lines in order to define better the molecular basis of HERG currents discovered in tumor cells. It was demonstrated previously (11, 30) that IHERG biophysical properties (rapid deactivation kinetics and strong dependence of the activation gate on depolarized Vm values) as well as biomolecular features (presence of multiple RNA bands revealed by Northern blot) in tumor cells are quite different from those displayed in the heart and in herg1-transfected cells. As reported here, all the tumor cell lines tested expressed the herg1 gene, whereas herg2 expression is limited to human retinoblastoma cells and herg3 to a human mammary carcinoma cell line. Whereas the expression of herg2 in retinoblastoma cells is in keeping with the well documented expression of erg2 in the retina of the rat (20) and quail (43), the presence of herg3 in a cell line of epithelial origin is quite surprising, because erg3 was first characterized as a nervous system-specific gene (20).
The following question thus arises: does the herg gene(s) expression in tumor cells represent 1) the re-expression of an embryonic gene or 2) the ectopic expression of a gene that is turned off in fully differentiated cell types? This is an important point for cancer researchers, as it has been well documented that neoplastic cells display biochemical and behavioral features of both embryonic, highly immature cells and novel, often metaplastic characteristics, completely altering the phenotype of the transformed cell. In neuroblastoma and rhabdomyosarcoma cells, herg1 expression appears to be the re-expression of an embryonic gene (see our results on quail embryos (43)). The type of expression for tumors of epithelial origins is less clear as neither herg1 nor herg3 genes are expressed in epithelial tissues in adults (20) or embryos (43).
The data reported here also demonstrate that tumor cell lines and primary human tumors express an alternative transcript of herg1, which results in an N-terminal truncated form. It is worth noting that in human tumors many cancer-associated genes are alternatively spliced (44). Although the function of most of these variants is not well defined, some of them have antagonistic activities related to cell death mechanisms. In many types of cancer and cancer cell lines, the ratios of the splice variants are frequently shifted toward the anti-apoptotic splice isoforms. In this regard, the herg1 gene may be among such genes that are alternatively spliced in neoplastic cells, raising interesting questions as to the different roles exerted by the full-length versus the spliced isoform in tumor cell establishment and maintenance.
These results also indicate that the truncated herg1 is the alternative transcript herg1b. The whole transcript of herg1b was cloned by RT-PCR and 3'-RACE in FLG 29.1 leukemia cells. This is the first report of the entire transcript in humans obtained thus far (GenBankTM accession number AJ512214). During the 3'-RACE cloning procedure, we also cloned another herg variant that resulted in the fusion of the herg1b exon with the hergUSO sequence. This transcript, lacking the 104 amino acid C-terminal domain necessary for channel recapitulation, is not expressed at the protein level on the plasma membrane and thus does not contribute to IHERG currents,3 but its putative role in tumor cells is now under investigation.
In contrast, the HERG1B protein is expressed on the plasma membrane and does form heterotetramers with the herg1 gene product in tumor cells, as demonstrated clearly by immunoprecipitation experiments reported in this paper. The herg1b transcript was first identified in heart (27, 28), but the corresponding protein is not expressed in adult hearts (29). Although it was suggested (29) that the protein could be expressed during development, the exact role of HERG1B in cell physiology is still unknown. Therefore, the demonstration that this channel protein is expressed in tumors, while confirming that tumor cells often express embryonic and/or alternatively spliced genes, opens new and interesting perspectives on the role of herg1 isoforms in tissues other than heart.
The expression of herg1b in neoplastic cells could explain both the peculiar pattern of mRNA expression and the biophysical features of HERG currents observed in tumor cells (11, 12, 30). HERG currents in such cells, especially in leukemia cell lines, display fast deactivation kinetics that may be attributable to expression of an N-terminal truncated HERG in these cells (30). As described previously (27, 28), the current encoded by herg1b displays most of the above biophysical features when expressed in oocytes.
Further experiments performed on synchronized cells show that whereas the truncated HERG1B form is up-regulated during the S phase, the full-length HERG1 protein increases its expression on the plasma membrane during the G1 phase. These results give a molecular dimension to the previously demonstrated variations of HERG activation curves, as well as of Vm during cell cycle progression of neuroblastoma cells (12). This result is also in keeping with other reports demonstrating cell cycle modulation of K+ channel expression (1), and in general with the reported link between K+ channels and cell cycle progression (45, 46). In different models (1) it has been reported that an increase in K+ channel expression and activity occurs at the G1/S boundary and that such an increase is necessary for cells to traverse the cell cycle. In HERG-bearing tumor cells, such as SH-SY5Y, an increase in the HERG1B/HERG1 ratio on the plasma membrane occurs as cells proceed through the S phase. This increase could account for the depolarization of Vm occurring during S phase progression of neuroblastoma cells, as reported previously (12), and the necessity of HERG channel activity for proliferation. This is demonstrated by impaired neuroblastoma and leukemia (41) cell proliferation in the presence of HERG-specific inhibitors. In other words, a tightly clamped Vm value is required for each cell cycle phase and can be obtained by alternatively switching the above-mentioned ratio; the Vm oscillations are apparently necessary for neuroblastoma cell cycling, so that cell proliferation stops either when the full-length HERG1 isoform is turned on and Vm hyperpolarizes (see RA-treated cells) or when the HERG isoforms-based clock is impaired by totally blocking HERG currents.
Finally, the truncated HERG1B isoform lacks the PAS domain, an oxygen-sensing domain of basic helix-loop-helix proteins, like HIF-1. The latter is a transcriptional activator that is up-regulated by hypoxia and is responsible for gene activation under hypoxic conditions (47). It is worth noting here that hypoxia is a main determinant of tumor progression and is currently regarded as a major hindrance to cancer therapy (48, 49). The ability of tumor cells to express two types of HERG proteins, one endowed with and the other lacking the PAS domain, could be an advantage for cancer growth and progression. In hypoxia, cells could thus sense the decreased oxygen tension by PAS, lowering the HERG1B/HERG1 ratio, thus leading to a shifting of the activation curve of HERG currents and to hyperpolarize Vm, limiting K+ loss (50). This could permit the cell to survive in G1 without entering into the apoptotic pathway, because K+ efflux is recognized as one of the earliest events in cells undergoing apoptosis (51). When the oxygen supply is restored and/or growth factors are produced, HERG-bearing tumors could undergo a remodeling of their HERG channels on the plasma membrane, increasing the HERG1B/HERG1 ratio, thus clamping Vm to depolarized values compatible with sustained cell growth.
Data presented in this paper, and in particular the demonstration of
protein expression of the herg1b transcript, provide a novel
perspective for a therapeutic approach to control HERG-expressing human
primary tumors like endometrial cancers (14), acute myeloid leukemias
(41), astrocytomas,4 and
colo-rectal cancers.5 Various
strategies (specific drugs or specific antisense oligonucleotides) could be designed to block specifically the altered HERG currents in
tumors without affecting the Ikr currents found in cardiac myocytes or other non-transformed cell types.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. E. Gherardi (MRC Centre, Cambridge, UK) and Dr. R. T. Wymore (University of Tulsa, Oklahoma) for reading the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Associazione Italiana Contro le Leucemie (Firenze) (to A. A.), Associazione Italiana Contro le Leucemie Comitato 30 ore (to A. A.), from the Ministero dell'Università e Ricerca Scientifica e Tecnologica (MURST, Cofin `99 and Cofin `01) (to A. A.), from the Associazione Italiana per la Ricerca sul Cancro (to M. O.), and from Ente Cassa di Risparmio di Firenze (to M. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ512214.
** To whom correspondence should be addressed: Dept. of Experimental Pathology and Oncology, Viale Morgagni 50, 50134 Firenze, Italy. Tel.: 39-055-4282326; Fax: 39-055-4282333; E-mail: annarosa.arcangeli@unifi.it.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M210789200
2 G. Hofmann, personal communication.
3 A. Arcangeli, A. Beechetti, L. Guasti, and O. Crociani, unpublished data.
4 A. Masi, personal communication.
5 E. Lastraioli, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: HERG, herg-encoded protein; herg, human eag-related gene; IHERG, HERG current; IRK, inward rectifier K+ channel; RPA, RNase Protection Assay; RT-PCR, reverse transcription PCR; hcyc, human cyclophilin gene; PBS, phosphate-buffered saline; HU, hydroxyurea; RA, retinoic acid; PI, propidium iodide; RACE, rapid amplification of cDNA ends; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PAS, periodic acid-Schiff.
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