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Molecular Oncology, Markers, Clinical Correlates |
Mayer Cancer Biology Research Laboratory, Department of Radiation Oncology, Stanford University School of Medicine, Stanford California 94305 [N. D., C. S., A. K., A. G.], and SRI International, Menlo Park, California 94025 [K. L., C. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hypoxia has also been shown to be a potent modulator of gene expression
in a wide variety of cell lines tested in vitro. Specific
protein accumulation (9
, 10)
, gene induction
(11)
, and gene repression (12)
have been
reported. Several transcription factors, AP-1 (13)
,
NF-
B (14)
, and
HIF-13
(11)
,
have been identified whose activity increases under hypoxic conditions.
HIF-1 is the transcription factor that responds most specifically and
robustly to changes in oxygen concentration. This transcription factor
is essential for development (8
, 15)
and is a heterodimer
composed of a hypoxia-responsive HIF-1
subunit and a non-hypoxia
responsive, constitutively expressed HIF-1ß subunit
(16)
. Under hypoxic conditions HIF-1 protein becomes
stabilized (17)
and binds with HIF-1ß to an HRE
containing the sequence 5'ACGTG(C/G)3' (18)
. Although
numerous HIF-1-responsive genes, such as VEGF
(19)
, glycolytic enzymes (20)
,
urokinase receptor (21)
, and endothelin
1 (22)
, have been reported in the literature, it is
still unclear whether any or all of these gene products are the
underlying reason why hypoxic tumors are more aggressive.
We hypothesize that determining the identity of hypoxia-induced genes would be critical for understanding the mechanism(s) responsible for the more aggressive nature of tumors that contain regions of hypoxia. In this communication, we describe the use of the RDA technique to identify hypoxia-induced sequence tags. Because the RDA technology has many potential technical pitfalls, the identity of each tag and the corresponding gene was confirmed by sequencing, and the hypoxia inducibility of each tag was confirmed by Northern blotting. Using these rigorous criteria, the screen identified two novel expressed sequences that we cloned in their entirety; these two novel genes are termed HIG1 and HIG2. HIG1 and HIG2 are also induced by the tumor microenvironmental stress of hypoglycemia, suggesting a coordinated response to both stresses. HIG1 and HIG2 may therefore represent a conserved mechanism for cells to respond to adverse microenvironmental stresses found within a tumor.
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MATERIALS AND METHODS |
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Hypoxic Conditions.
Cells were plated overnight in vented glass Petri dishes at 5 x
106 cells/100 mm dish. Hypoxic conditions were
generated by placing the dishes in an anaerobic chamber (Sheldon
Laboratories, Cornelius, OR) that was flushed with a gas mixture of
90% N2, 5% CO2, and 5%
H2. Any oxygen that was introduced into the
chamber was consumed over a catalyst with hydrogen. A monitoring oxygen
electrode was used to confirm an environment of 0.05% oxygen or less
during experimentation.
RDA Technique.
Briefly, the RDA technique (24)
was performed on
double-stranded cDNA that served as starting material for multiple
rounds of in vitro subtraction and amplification. The cDNA
was generated from mRNA isolated from control and 16-h hypoxia-treated
HCE.E6E7 cells. The cDNA populations were digested with the restriction
enzyme NlaIII and ligated to different double-stranded
linkers described in Table 1
(linker 1
and linker 2). The modified cDNA fragments were then individually
amplified using a single primer corresponding to the linker sequence
described in Table 1
(primer 1 and primer 2). The primer amplifying the
driver population of fragments contained a 5' biotin label. Three
micrograms of biotinylated, driver cDNA and 0.1 µg of tester,
nonbiotinylated cDNA were mixed together, lyophilized, resuspended in 2
µl of hybridization buffer (50 mM HEPES, pH
7.5, 10 mM EDTA, 1.5 M
NaCl, and 2% SDS), covered in mineral oil, denatured at 95°C for 10
min, slowly cooled to 68°C over 1 h, and kept at 68°C for 4
more hours to allow hybridization. The hybridized cDNA populations were
then diluted, mixed, and bound to 1 mg of M280 Dynal Strepavidin beads.
The biotinylated DNA and any hybrid DNA was then removed with a magnet.
The remaining, differentially expressed cDNA was reamplified using the
tester primer, and then the subtraction was repeated three more times.
The final cDNA population was digested with NlaIII and
cloned into the SphI site of pUC18 to generate
the library of enriched fragments.
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Construction of Epitope-Tagged HIG1 and
HIG2.
HIG1-HA and HIG2-HA were constructed by reverse
PCR. A (minus) primer was synthesized (Table 1
, hHIG1HA[rev] or
hHIG2HA[rev]) that hybridized to the carboxyl terminus of the ORFs.
It extended the coding sequence by removing the endogenous stop codon,
adding 36 nucleotides that code for 12 amino acids (the HA epitope),
followed by a new stop codon, and ending with an MluI site.
A second (plus) primer was synthesized (Table 1
, hHIG1HA[for], or
hHIG2HA[for]) that contained an MluI site, followed by a
region that hybridized to the beginning of the 3' untranslated region.
The plasmid containing the cloned gene in an expression cassette
(pEGFPN1 with the GFP removed; Clontech) was then
used as a template for PCR amplification using pfu
polymerase (Stratagene). The full-length linear molecule containing the
added sequences was then digested with MluI, ligated closed,
and used to transform competent bacteria.
Immunological Detection of Epitope Tags.
For immunohistochemical detection of HIG1HA and HIG2HA, cells were
grown on chamber slides and were transfected with the indicated
constructs using LipofectAMINE according to the instructions of the
manufacturer (Life Technologies, Inc.). After 48 h the transfected
cells were fixed in 2% paraformaldehyde, washed two times with PBS-T,
blocked for 1 h in PBS-T with 3% BSA, and incubated for 1 h
with anti-HA monoclonal antibody (Babco 101R) at 1:500 dilution in
PBS-T with 3% BSA. The anti-HA treated slides were washed three times
with PBS-T, incubated with fluoresceinated secondary goat antimouse
antibody (Vector), also in PBS-T-BSA, washed three times in PBS-T,
coverslipped with antifade solution (Vector), and visualized under
epifluorescence using a Nikon microphot fluorescent microscope.
For immunoblot detection of HIG1HA and HIG2HA, protein extracts were generated from cell populations transiently transfected with the indicated expression plasmids. Cell populations were harvested and resuspended in PBS containing the protease inhibitor PMSF, 1.0 mM of the phosphatase inhibitor Na3VO4, and 1.0 mM of the kinase inhibitor NaF. Twenty-five micrograms of the extracts were electrophoresed on a 15% tricine gel and electrotransferred to the polyvinylidine difluoride membrane. The membranes were then blocked with PBS-T containing 5% milk for >1 h, incubated with anti-HA antibody at a 1:2000 dilution for 1 h in PBS-T milk, washed three times in PBS-T, incubated with horseradish peroxidase-conjugated goat antimouse antibody at a 1:2500 dilution in PBS-T milk, washed three times in PBS-T, and visualized with enhanced ECL (Amersham, Rockford, IL) on the Storm 860 (Molecular Dynamics).
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RESULTS |
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Because some of the genes are represented multiple times in this
library, the 500 tags represent fragments of 21 genes that are present
more than one time and 168 genes that are represented only once (Table 2)
. The two most frequently occurring
genes are HIG1 and HIG2. All of the clones
represented more than one time that did not contain a highly repetitive
element were tested by Northern blot for induction by hypoxia as well
as by hypoxia and reoxygenation in SiHa cervical carcinoma cells.
Representative Northern blot analysis is shown in Fig. 1
to demonstrate the kinetics of
induction for each of the induced genes. It is interesting to note that
approximately one third of the tags in Table 2
(7/19) were not induced,
so it becomes clear why it is necessary to test each probe by Northern
analysis. Although acetoacetyl-CoA thiolase sequence tag is listed as
induced, the reported, major RNA (1.8 kb) for the gene does not change.
However, there is a larger, hybridizing RNA species (4.2 kb) that is
induced after 24–48-h hypoxia (data not shown). The Northern blot for
annexin V is shown from HCE.E6E7 cells because annexin V is weakly
induced in SiHa cells.
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that are not shown in Fig. 1
had only moderate hypoxic
induction (2–3-fold). There are three genes identified in this group
that have been previously reported to be hypoxia-inducible:
GAPDH (26)
, tissue factor
(27)
, and thioredoxin (28)
.
Because HIG1 and HIG2 represent two novel genes
whose functions are unknown, we investigated these genes in more
detail. We first examined the expression of HIG1 and
HIG2 in a series of cervical cancer cell lines under oxic
and hypoxic conditions in vitro (Fig. 2)
. Although HIG1 is induced
moderately within 2 h of hypoxia in all of the cell lines tested,
it only remains elevated only in the SiHa cells. HIG2 is
more consistently induced from low basal levels in all of the cervical
cancer cells tested. The major HIG2 mRNA species is 1.4 kb
in length, but there are two other mRNA species of minor abundance (8.0
and 9.0 kb) that are induced with identical kinetics to the major
species (data not shown). The hypoxic induction of HIG1 and
HIG2 in vivo was also tested in tumor xenografts
generated from the C33a cell line by Northern blot analysis of total
tumor RNA. We compared untreated xenografts to xenografts that were
made hypoxic by treatment of the host animal with FAA 24 h prior
to explantation and RNA isolation (Fig. 2)
. We chose to examine
expression changes after 24 h because this is the time shown to
result in the most dramatic ablation of the tumor vasculature before
causing extensive parenchymal necrosis. FAA treatment resulted in
increased tumor hypoxia as measured by Eppendorf electrode (data not
shown) and increased HIG1 and HIG2 expression by
1.2- and 2.4-fold, respectively. The moderate level of HIG1
induction in vivo is not unexpected, because of the in
vitro data. The portion of the human gene used as a probe in these
experiments has low homology with mouse RNA and under the conditions
used did not cross-hybridize.
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. Both ORFs encode small peptides
(93 and 63 amino acid residues). Both peptide sequences were run
through NCBI pBLAST, and no existing protein sequences of significant
similarity were found. No functional motifs were identified in either
sequence using world wide web-based search programs Prodom-Blast at
Institut National de la Rescherche
Agronomique5
or Propsearch at
European Molecular Biology
Laboratory-Heidelberg.6
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Figure 5
shows Northern blot analysis of
RNA isolated from C33a cells exposed to these stresses. HIG1
is poorly responsive to hypoxic stress over this time course (Fig. 2)
.
HIG2 is induced strongly by hypoxia, the hypoxia-mimetic
stress inducer deferoxamine, as well as glucose deprivation. UV
light seemed to have little effect upon either HIG1 or
HIG2 expression. In contrast, although ionizing radiation
did not change HIG1 expression levels, it did result in a
moderate 2.5-fold induction of HIG2 by 24 h. The
similarities in the pattern of stress responsiveness of HIG2
and that of the HIF-responsive VEGF gene suggest that
HIF-1 may be important in HIG2 expression.
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, HIG1; Fig. 5F
, HIG2). Extracts of these cells were also
examined by immunoblot for the protein size (Fig. 5
A, HIG1,
5D, HIG2). Expression of a peptide is detected
only in the cells transfected with plasmids expressing the tagged
protein(s).
Immunoreactive material was detected in punctate pattern throughout the
cytoplasm for HIG1, suggesting a vesicular or mitochondrial
location. Immunoreactive material was found in a more diffuse,
cytoplasmic localization for HIG2. It is difficult to draw
too many conclusions from the cellular localization patterns of
proteins that are overexpressed. Forced overexpression of some proteins
can overwhelm normal trafficking patterns leading to aberrant
localizations. Cellular morphology can be delineated by viewing Fig. 5, B and D
, which represent the same fields seen in
Fig. 5, C and F
, but visualized under UV to
excite the DAPI-stained nuclei. Because HIG2 appeared on the
immunoblot as multiple bands, we hypothesize that this might represent
multiple processed forms of the protein, producing altered migration.
Interestingly, hypoxic treatment did not alter the protein quantity or
electrophoretic mobility of the heterologously expressed proteins in
C33a cell populations transfected with CMVHIG1HA or CMVHIG2HA and
treated with hypoxia for 6 h (Fig. 5, A and D)
.
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DISCUSSION |
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The Ku(70) gene product has been shown to participate in the repair of DNA double-strand breaks and could hypothetically respond to hypoxia-induced DNA damage (29) if it occurs. Another possibility is that hypoxia-stimulated Ku(70) expression could act as a tumor suppressor (30) as has been reported. Regardless of the reason, if low oxygen leads to Ku(70) induction, one side effect could be increased resistance to DNA-damaging agents or ionizing radiation.
Hypoxia has been shown to induce the expression of rate-limiting enzymes necessary for increased glycolysis in the absence of oxidative phosphorylation (20) . One additional product of glycolysis is the generation of (reduced) NADH. The major generation of NADH during glycolysis is from the activity of GAPDH. The induction of GAPDH therefore serves two functions: (1) to contribute to increased glycolysis, and (2) to increase the production of reduced NADH. If hypoxic damage were mediated through a redox imbalance, then it would also be reasonable to induce a system for titrating the extra reducing/oxidizing equivalents. These extra reducing equivalents also could be used by several different cellular processes. For instance, it has been shown that thioredoxin can use NADH as a proton donor to activate the transcription factor AP-1 (31) or the estrogen receptor (32) . To generate a large cellular pool of NADH, it might be necessary to synthesize more NAD. One of the precursors for NAD is adenine, and the rate-limiting step in the de novo generation of purines is PRPP synthase. Thus, PRPP synthase induction by hypoxia may be a physiological response to a redox imbalance.
Annexin V and lipocortin 2 code for two family members of a group of cell surface calcium-binding proteins. Both molecules have been shown to play a role in the regulation of the fibrinolytic activity of plasmin (33) . These gene products also decrease cell motility in vitro (34) . Thus, as the cell surface properties of hypoxic cells could regulate adhesion and cell-cell connections in a tumor, the annexins, in concert with hypoxia-responsive proteases such as calpain (35) , could impact tumor invasiveness and metastatic potential.
Wound healing, clot formation, and revascularization rely on delicately balanced factors, and one of the most potent inducers of angiogenesis is tissue hypoxia. It is not clear what the signaling mechanism is that leads to vessel generation, but many hypoxia-responsive genes are involved, such as VEGF (19) and endothelin 1 (22) . Hypothetically, hypoxia could act as a regulator of fibrinolysis by modification of plasmin activity through annexin induction (33) . Additionally, tissue factor has been shown to regulate both the generation of fibrin (36) and the angiogenic activity of VEGF (37) . Thus, tissue factor expression in response to hypoxia could be important in regulating the function of the vasculogenic factors of the tumor and as such could influence the growth rate of the tumor.
There are several reasons why we did not isolate all of the known hypoxia-inducible genes (such as VEGF) in this series of experiments. The primary reason is the nonrandom distribution of cleavage sites for the restriction enzyme NlaIII used for digestion of cDNAs prior to linker ligation. Those genes, such as VEGF, with NlaIII restriction sites organized in a nonrandom manner would generate fragments outside the 100- to 300-bp size that is most efficiently hybridized with the current protocol. The unlinked fragments, or the very large or very small fragments would be lost through the multiple rounds of subtraction and amplification. Another possibility is that additional bona fide hypoxia-inducible genes exist in the list of single hits from the 500 sequences. We chose to stop sequencing clones at 500 because we feel that this number gives good coverage to the complexity of our RDA library. We make this statement after comparing the diversity of the first 100 clones that were sequenced to the diversity of the next 400 clones that were sequenced.
Human HIG2 has a high fraction of serine and threonine
residues, 8 and 6 residues, respectively, of the total of 64 amino acid
residues (Fig. 3)
. Serine 41 conforms to the consensus PKC
phosphorylation site. Although HIG2 migrates as a complex
pattern of proteins, we could not detect a molecular weight shift of
the protein with in vitro treatment of cellular extracts
with nonspecific phosphatase (data not shown). It is therefore unclear
what the multiple forms of the protein represent, although the major
form of the expressed ORF does migrate with a mobility consistent with
the predicted size. However, the modifications do not seem to be
stress-inducible, and the uniform cytoplasmic distribution of the
epitope-tagged HIG2 does not provide clues to its function.
Taken together, these data support the hypothesis that there exists a cellular response to hypoxic stress that is regulated at the transcriptional level. This response is evolutionarily conserved in rodents and humans and consists of the coordinated regulation of many genes by HIF-1-dependent mechanisms as well as HIF-1-independent mechanisms so that the cell can survive in this adverse environment. The byproducts of this epigenetic response to low oxygen results in a tumor that is more resistant to conventional therapy and is more likely to invade or metastasize. The genes described in this report therefore represent potential new hypoxia-regulated proteins that can influence clinical outcome.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grants R01-CA-64489 and
CA-73807, and in part in collaboration with Varian Biosynergy. N. D.
was funded by NRSA individual fellowship CA88877. 
2 To whom requests for reprints should be
addressed, at Room GK220 CBRL, Department of Radiation Oncology,
Stanford University School of Medicine, Stanford CA 94305. Phone:
(650) 725-6418; E-mail: ndenko{at}cmgm.stanford.edu
3 The abbreviations used are: HIF-1,
hypoxia-inducible factor one; CMV, cytomegalovirus; EST, expressed
sequence tag; FAA, flavone acetic acid; HA, hemaglutinin; HCE, human
cervical epithelial cells; HIG1 and HIG2,
hypoxia-inducible gene 1 and gene 2, respectively; HPV, human papilloma
virus; HRE, hypoxia-responsive element; NCBI, National Center for
Biotechnology Information; ORF, open reading frame; PBS-T, PBS-0.2%
Tween 20; RDA, representational difference analysis; VEGF, vascular
endothelial growth factor.
4 The HIG1 complete sequence can be
found at NCBI GenBank accession no. AF145385 and HIG2 at
no. AF144755.
5 Address:
http://www.protein.toulouse.inra.fr/prodom/doc/prodom.html.
6 Address: http://www.embl-heidelberg.de/prs.html.
Received 8/27/99; revised 11/ 8/99; accepted 11/12/99.
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REFERENCES |
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