From the Department of Pathology and Comprehensive
Cancer Center, The Ohio State University, Columbus, Ohio 43210 and
§ Department of Immunology, Roswell Park Cancer Institute,
Buffalo, New York 14263
Received for publication, January 29, 2003
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ABSTRACT |
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Both viruses and tumors evade cytotoxic T
lymphocyte-mediated host immunity by down-regulation of antigen
presentation machineries. This can be achieved by either
down-regulation of transcription of antigen presentation genes or
posttranslational inactivation of proteins involved in antigen
presentation. In this study, a major histocompatibility complex (MHC)
class I-deficient melanoma cell line, SK-MEL-19, was found deficient in
the expression of the transporter associated with antigen processing
(TAP)-1 mRNA even after IFN- Recent studies demonstrate that patients with malignant melanoma
often have a high number of cytotoxic T lymphocytes specific for
melanoma-associated antigens (1, 2). The co-existence of T cells and
tumor cells even in the draining lymph nodes suggests that the tumors
were able to evade destruction by host cytolytic T lymphocytes.
Accumulating evidence supports the notion that both malfunction of T
cells and down-regulation of antigen presentation machinery in tumors
can be responsible for tumor evasion of host immunity (1-6). In fact,
a high proportion of malignant tumors, including melanoma, have
severely depressed cell surface expression of class I HLA antigens (7),
the target molecules that present tumor antigenic peptide to cytolytic
T lymphocytes. Understanding the mechanisms underlying the T-cell
malfunction or antigen presentation defects may thus provide insight
for immunotherapy of melanoma and other cancers.
Optimal cell surface expression of HLA molecules requires the
coordinated expression of several genes, such as transporters associated with antigen processing
(TAP)1-1/2,
low molecular weight peptide (LMP)-2/7,
and tapasin, as well as HLA class I heavy chain and
Cell Lines and Antibodies--
Human melanoma cell lines 1195, 1102, and SK-MEL-19 were cultured as described previously (15). The
breast cancer cell line SK-BR-3 was obtained from ATCC (HTB-30; ATCC,
Manassas, VA). All cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). For induction of HLA class I
expression, cells were cultured in medium supplemented with recombinant
human IFN- Flow Cytometry--
Cell surface HLA class I expression was
examined by flow cytometry as described previously (4). Briefly, viable
cells were incubated with PE-conjugated mouse IgG1 and PE-conjugated
anti-HLA-A, -B, and -C antibody at 4 °C for 2 h. After three
washes with phosphate-buffered saline containing 1% fetal calf serum,
cells were fixed with 1% paraformaldehyde and examined by flow cytometry.
Northern Blot--
Cells were either treated with IFN- Generation of TAP-1 cDNA Constructs and Stable
Transfection--
The human small cell carcinoma H146 cell line
(provided by Dr. N. P. Restifo; National Cancer Institute, Bethesda,
MD) was incubated with IFN- Southern Blot--
Genomic DNA was isolated from SK-MEL-19
cells, SK-BR-3 cells, and HeLa cells. Genomic DNA (20 µg) was
digested with AflIII (Invitrogen) and separated in 0.8%
agarose gel. The TAP-1 promoter probe was made by PCR from
normal human lymphocyte genomic DNA with sense primer
5'-TCCCGCCTCGAGCATCCCTGCAAGGCA-3' and antisense primer
5'-TGCAGTAGCCTGGTGCTATCCG-3'. Probes were labeled as described above.
Generation of Luciferase Reporter Constructs and Assay for
Promoter Activity--
The TAP-1 promoter was amplified
from SK-MEL-19 cell genomic DNA by PCR using the following primers:
hTAP1.Pr1, 5'-GCTCTAGATGGCACTCGGACGCCGTC-3'; and hLMP2.Pf1,
5'-GCTCTAGACCCTGCAAGGCACCGCTC-3'. The PCR products were subcloned
using the Zero Blunt TOPO PCR cloning kit (Invitrogen) and then cloned
into pGL2-basic vector (Promega, Madison, WI) at XhoI and
HindIII sites. All constructs were confirmed by DNA sequencing. Expression level of the firefly luciferase from the pGL2
constructs (basic, SV40, pTAP1/T, and pTAP1/G) was normalized to the
internal control pRL-SV40 Renilla luciferase level. Results were shown as the fold increase compared with the pGL2-basic. The dual
luciferase assay was carried out according to the manufacturer's instructions (Promega).
Nuclear Run-on Assay--
The assay was performed as described
elsewhere (16). Briefly, nuclei were extracted from 107 to
108 SK-MEL-19 cells treated with or without 1000 units/ml
IFN- Restriction Fragment Length Polymorphism--
Primers hTAP1CE7.f
(5'-GCACCCCTCGCTGCCTACCCAGTGGTCT-3') and hTAP1CE7.r
(5'-TACAGGGAGTGGTAGGTTGTACCTG-3') were used to amplify from the genomic
DNA the region of TAP-1 exon 7 where the single-nucleotide deletion resides. The region was also amplified from cDNA using primers hTAP1CE7.f and hTAP1CE7.r PCR products were separated by gel
electrophoresis and purified using Qiagen gel extraction kit (Qiagen,
Valencia, CA). The purified PCR products were incubated with
BslI (New England Biolabs, Beverly, MA) at 55 °C
overnight and then separated in 5% agarose gel.
Generation of TAP-1 cDNA and Tet-Off SK-MEL-19 Cell
Lines--
Wild type TAP-1 cDNA was cloned from the
human small cell carcinoma H146 cell line as described above. TAP-1
D1489 was generated by PCR using the total cDNA from the SK-MEL-19
cells as the template. Site-directed mutagenesis by overlapping PCR was
performed to make TAP-1 Del3 cDNA. After confirming their
sequences, all three TAP-1 cDNAs were inserted into the
multiple cloning site of the pBI-EGFP vector
(Clontech) by blunt-end ligation. The three
constructs, pBI-EGFP-TAP1, pTet-Off (Clontech), and
pcDNA3.1/Hyg(+) (Invitrogen), were co-transfected into SK-MEL-19
cells using FuGENE 6 transfection reagent (Roche Molecular
Biochemicals). Stably transfected cell clones were selected in 96-well
plates in Dulbecco's modified Eagle's medium supplemented with 0.5 mg/ml hygromycin (Invitrogen). To confirm the efficiency of the Tet-Off
construct in the tumor cell line, the green fluorescence
protein-positive cell clones were treated with 1 µg/ml
tetracycline (Roche Molecular Biochemicals) for 24 h. The total
RNA was extracted from the cell clones with or without tetracycline
treatment. The cell clones in which TAP-1 mRNA
expression was inhibited by at least 95% by tetracycline were used for
the study of mRNA stability.
RNase Protection Assay--
The TAP-1 cDNA
construct used to make the TAP-1 antisense transcript was
the same as the one used for the Northern blot. The GAPDH
cDNA fragment was generated by PCR using the primers hGAPDH.f (5'-TGAGAACGGGAAGCTTGTCATCAA-3') and hGAPDH.r
(5'-CAGCCTTCTAGATGGTGGTGAAGA-3'). The EGFP cDNA fragment was also
generated by PCR using the primers EGFP.f
(5'-TCCAGCAGGATCCTGTGATCGCGCT-3') and EGFP.r
(5'-ACCTACGGCCTCGAGTGCTTCAGCC-3'). The antisense probes were made using
the Riboprobe in vitro transcription system (Promega). The
RNase protection assay was conducted with the RPA III ribonuclease
protection assay kit (Ambion) according to the instruction manual.
After separation of protected fragments on a 6% sequencing gel,
signals were quantified by phosphorimaging (Amersham
Biosciences). The TAP-1 signal intensity was
normalized by that of the GAPDH signal, which served as a
loading control. The percentages of the amount of remaining
TAP-1 mRNA at different time points after tetracycline
was added compared with time 0 were calculated.
Down-regulation of TAP-1 mRNA by a Posttranscriptional Mechanism in
Melanoma Cell Line SK-MEL-19--
Three human melanoma cell lines
(1102, 1195, and SK-MEL-19) were examined by flow cytometry for their
cell surface HLA class I expression with or without IFN-
Because optimal cell surface HLA class I expression requires the
coordinated expression of multiple genes, including
TAP-1/2, LMP-2/7, and
It has been known that TAP-deficient cells can express HLA class I
after transfection with the TAP-1 or TAP-2 gene
(17-19). To test whether the lack of TAP-1 expression was
responsible for the barely detectable expression of HLA class I antigen
on the surface of SK-MEL-19 cells, we transfected the cells with
TAP-1 cDNA. As shown in Fig. 1c, the
TAP-1 cDNA-transfected SK-MEL-19 cells expressed
significant levels of HLA class I antigen even before
IFN-
Because the TAP-1 expression was low at the mRNA level,
we hypothesized that the TAP-1 down-regulation was caused by
defective transcription or malfunction in RNA metabolism. The
TAP-1 expression is under the control of a bidirectional
promoter, as characterized by Wright et al. (20). We cloned
and sequenced the 593-bp TAP-1 promoter from SK-MEL-19
cells. In comparison with the published sequence (20), a
single-nucleotide G
We therefore performed a nuclear run-on assay to directly evaluate the
transcription of the TAP-1 gene. LMP-2
transcription, which is under the control of the same bidirectional
promoter, was also evaluated. As shown in Fig. 2c,
TAP-1 was transcribed at high levels in SK-MEL-19 cells
under basal conditions, although IFN- A Single-nucleotide Deletion Leads to Accelerated Decay of TAP-1
mRNA--
A major mechanism responsible for posttranscriptional
regulation of mRNA is RNA degradation, which can be prevented by
CHX, a protein synthesis inhibitor of mammalian cells. It is well
established that the turnover of mRNA is closely linked to the
translation process and that blocking translation can stabilize
mRNA, especially those mRNA with short half-lives
(21-23). To test whether accelerated RNA degradation is responsible
for the lack of TAP-1 in SK-MEL-19 cells, we treated the
SK-MEL-19 and control HeLa cells with CHX after incubation with or
without IFN-
The rapid degradation of TAP-1 mRNA can be due to a
genetic lesion in the TAP-1 gene. Alternatively, it is
possible that the tumor cell line expresses factors that can cause
TAP-1 mRNA degradation. The successful rescue of cell
surface HLA class I antigen expression by wild type TAP-1 in
SK-MEL-19 cells favors the first hypothesis because a wild type
cDNA can be expressed in the tumor cell line. As the first step to
test this hypothesis, we cloned TAP-1 cDNA from
SK-MEL-19 cells that were treated with both IFN-
Premature termination codons resulting from frameshift mutation or
nonsense mutation have been shown to interfere with the metabolism of
many different mRNAs in mammalian cells, leading to
nonsense-mediated altered RNA splicing, such as exon skipping and
intron retention and/or NMD (21-23). Alternatively, a mutation may
disrupt a cis-element that is necessary for mRNA stability and
thereby cause RNA decay. To differentiate the two possible mechanisms,
we designed another TAP-1 mutant (TAP-1 Del3) that has an
in-frame 3-nucleotide deletion at the same position as TAP-1 D1489
(Fig. 5a) and compared the
mRNA half-lives of TAP-1 WT, TAP-1 D1489, and TAP-1 Del3. If the
degradation of TAP-1 D1489 is via the NMD pathway, then the half-life
of the TAP-1 Del3 message should be comparable with that of TAP-1 WT.
Otherwise, if the message of TAP-1 Del3 is comparable with or even more
unstable than that of the TAP-1 D1489, then it is the deletion itself, but not the resulting premature termination codons, that leads to the
accelerated decay of TAP-1 mRNA in SK-MEL-19 cells. The Tet-Off gene expression system was adapted (Fig. 5a) to
compare the stabilities of various TAP-1 mRNAs. We first
selected the SK-MEL-19 transfectants with an induction ratio (Tet 0/Tet
24 h) of 20 or greater to test mRNA half-lives. This ensured
that more than 95% of transcription was blocked by the tetracycline after 24 h. The selected cell clones were then treated with 1 µg/ml tetracycline for different lengths of time before the total RNA
was harvested. The amounts of the mRNA were quantified by phosphorimaging. In addition, the intensity of the TAP-1
signal was normalized to that of GAPDH before the percentage
of remaining mRNA was calculated. Four experiments were conducted,
and an average percentage value was used to derive the mRNA
half-lives (t1/2) via regression analysis. As shown
in Fig. 5, b and c, with a
t1/2 of 7.2 h, the TAP-1 WT mRNA is
considerably more stable than that of the TAP-1 D1489
(t1/2 = 3.5 h). However, the mRNA derived
from TAP-1 Del3 (t1/2 = 2.7 h), which has an
in-frame 3-nucleotide deletion, was degraded at least as fast as that
of TAP-1 D1489. Because the TAP-1 Del3 mRNA has no premature
termination codon downstream to the deletion, the accelerated decay in
mutant TAP-1 mRNA is most likely through mechanisms
other than NMD. It is more likely that the mutation disrupts a
cis-element critical for the stability of TAP-1 mRNA. Whereas few cis-elements that help to stabilize mRNA have been reported, at least two have been reported by others (24, 25) and our
group (26). Preliminary analysis showed no similarity between the
region surrounding the mutation and the previously reported
cis-element.
Nevertheless, the NMD is a well-conserved cellular surveillance
mechanism. Whereas our work revealed a non-NMD mechanism for TAP-1 mRNA degradation, it is still possible that
mRNA derived from the endogenous mutant TAP-1 gene can
also be degraded by NMD. It has been shown recently that several
criteria have to be met for the pathway to degrade a premature
termination codon-containing message. First, at least one downstream
spliceable intron is required for optimal NMD (27-29). The intron is
thought to help recruit NMD factors, such as hUpf3, to the mRNA via
the spliceosome (30-32). Second, the premature termination codon
should be at least 45-55 nucleotides away from the next spliceable
intron (27, 28). The lack of introns in our constructs may have
prevented us from revealing NMD in TAP-1 mRNA
degradation. However, intronless premature termination codon-containing
HEXA mRNA was shown to be subject to NMD, although at a lower
efficiency than that seen when multiple downstream introns are present
(33). In preliminary studies, when we made pBI-EGFP/TAP1 constructs
with intron 7 or 8, the results also failed to support a role for NMD
in degradation of the mutated TAP-1 mRNA (data not shown).
Posttranscriptional regulations of other genes involved in
antigen presentation have been reported previously (34). The increased
turnover of HLA-C heavy chain mRNA has been suggested to contribute
to the low level of HLA-C surface expression (34). Our work shows
that mutations in the TAP-1 gene in a tumor cell line can
modulate its mRNA stability. This mechanism may be exploited by
tumors to evade host immunity.
stimulation, despite its active transcription of the TAP-1
gene. This abnormality was caused by a single-nucleotide deletion
at position +1489 of the TAP-1 gene and was corrected by cycloheximide, which inhibits RNA degradation. Using an inducible Tet-Off system, we demonstrated that deletion of the nucleotide resulted in a >2-fold decrease in the half-life of TAP-1
mRNA. However, the decrease of the half-life of TAP-1
mRNA is not mediated by nonsense-mediated mRNA decay because
deletions of two additional nucleotides in the region, which corrected
the nonsense mutation, did not restore TAP-1 mRNA
stability. To our knowledge, this is the first evidence that the
degradation of mRNA of an antigen presentation gene is involved in
HLA class I down-regulation in malignant cells.
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2-microglobulin (
2M). In cases of both
tumorigenesis and viral infection, expression of these genes and the
function of the encoded proteins are often impaired (8-10). The
mechanisms for such down-regulation have been studied extensively.
Theoretically, gene expression can be modulated by transcriptional,
posttranscriptional, translational, and posttranslational mechanisms.
The mechanisms that have been shown to underlie the antigen
presentation abnormalities are transcriptional suppression of antigen
presentation genes and/or functional inactivation of their gene
products, either by missense mutation or by protein-protein interactions (11-14). Here we show that actively transcribed
TAP-1 mRNA in the melanoma cell line SK-MEL-19 is
rapidly degraded even after stimulation with IFN-
. Cloning and
sequencing analysis have revealed a single-nucleotide deletion at
position +1489. This mutation results in substantial reduction of the
stability of TAP-1 mRNA by mechanisms unrelated to
nonsense-mediated mRNA decay (NMD). These results reveal a new
potential mechanism for tumor evasion of host T-cell recognition.
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(1000 units/ml; R&D Systems, Minneapolis, MN).
PE-conjugated anti-HLA-A, -B, and -C antibody (clone G46-2.6) and
isotype control PE-conjugated mouse IgG1 were purchased from BD
PharMingen (San Diego, CA).
(R&D
Systems) at 1000 units/ml for 48 h or left untreated. For
cycloheximide (CHX; Sigma) treatment, SK-MEL-19 cells were cultured
with IFN-
at 1000 units/ml for 48 h, and then CHX was added to
the cells for a final concentration of either 5 or 10 µg/ml,
respectively, for up to 16 h. Total RNA was isolated using TRIzol
reagent (Invitrogen). Hybridization conditions followed the
instructions of the Northern hybridization kit (Eppendorf Scientific,
Westbury, NY). The cDNA probes for TAP-1,
TAP-2, LMP-2, LMP-7, HLA class I heavy
chain, and
2M were made from PCR products using primers
listed previously (11). A human splenocyte cDNA library from
Invitrogen was used as the template for PCR reactions. All PCR products
were subcloned into pBluescript vector (Stratagene, La Jolla, CA) and
sequenced and confirmed to be identical to published sequences. The
probes were labeled with [
-32P]dCTP (PerkinElmer Life
Sciences) using the DECAprimeTM II kit (Ambion,
Austin, TX).
at 1000 units/ml for 48 h. Total
RNA was isolated as described above. Reverse transcription was done
using the SUPERSCRIPT First-Strand cDNA Synthesis System
(Invitrogen). TAP-1 cDNA was amplified by PCR in three
fragments. Primers were as follows: hTAP.f1,
5'-GCGGCCGCTTTCGATTTCGCTTTC-3'; hTAP.r1, 5'-TGCAGTAGCCTGGTGCTATCCG-3'; hTAP.f2,
5'-CTTGCCTTGTTCCGAGAGCTGA-3'; hTAP.r2, 5'-CTCGTTGGCAAAGCTTCGAAC-3';
hTAP.f3, 5'-CGGCCATGCCTACAGTTCGAAG-3'; and hTAP.r3,
5'-ATAAATATCAAGAACCTACAGGG-3'. The three fragments were cloned
into pBS-KS vector (Stratagene) at NotI/SmaI,
SmaI/HindIII, and
HindIII/XhoI sites, respectively, and sequenced
to confirm that the cDNA has a wild type sequence. When SK-MEL-19
cells grew to 70% confluence, 0.2 µg of pcDNA3.1/Hyg(+) vector
(Invitrogen) and pcDNA3.1/Hyg(+) vector with wild type
TAP-1 cDNA insert were respectively transfected into
each well of a 24-well plate, using 6 µl of FuGENE 6 transfection
reagent (Roche Molecular Biochemicals) according to the manual.
48 h later, the transfected cells were replated into 96-well
plates and cultured in Dulbecco's modified Eagle's medium and in the
presence of 0.5 mg/ml hygromycin B (Invitrogen). Single cell clones
were selected for further culture and analyzed for HLA class I antigen expression.
. The transcripts were labeled in vitro with 40 nM biotin-16-UTP (Roche Molecular Biochemicals) in the
presence of 3.75 mM ATP, GTP, and CTP; 25 mM
Tris-HCl; 12.5 mM MgCl2; and 750 mM
KCl. cDNA fragments of LMP-2 and GAPDH were
amplified by PCR from cloned cDNA constructs (11). TAP-1
cDNA fragment was amplified by PCR from cloned cDNA constructs
using primers hTAP1.f1 and hTAP1.r1 described above. The
pcDNA3.1/Hyg(+) vector was linearized with HindIII. All
the DNA was immobilized on nitrocellulose membrane using S&S Minifold
II slot blot apparatus according to the manual (Schleicher & Schüll). Hybridization conditions were as described previously
(16), and the biotin-labeled transcripts were detected using
streptavidin-alkaline phosphatase conjugate (Roche Molecular Biochemicals) and CDP-star Ready-To-Use with Nitro-Block-II reagent (Tropix, Bedford, MA).
RESULTS AND DISCUSSION
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ABSTRACT
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stimulation. A PE-conjugated anti-human HLA-A, -B, and -C antibody was
used to detect all HLA class I alleles, and a PE-conjugated mouse IgG1
was used as isotype control. As shown in Fig.
1a, 1102 and 1195 cells had
significant HLA class I that was further up-regulated by incubation
with 1000 units/ml IFN-
for 3 days. Confirming previous studies
(15), we found that SK-MEL-19 cells had no cell surface HLA.
Surprisingly, whereas other melanoma cell lines up-regulated their cell
surface HLA in response to IFN-
, very little HLA class I antigen
could be found on the SK-MEL-19 cells even after IFN-
-treatment.
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Fig. 1.
Deficiency of surface HLA class I expression
in melanoma cell line SK-MEL-19 was due to the TAP-1
down-regulation. a, HLA class I expression in
three melanoma cell lines, SK-MEL-19, 1102, and 1195. Bold
black lines depict the staining by PE-conjugated anti-human HLA-A,
-B, and -C antibody in untreated cells; dotted lines
represent the staining by PE-conjugated mouse IgG1 as isotype control;
and red lines represent anti-HLA-A, -B, and -C antibody
staining after stimulation with 1000 units/ml IFN- for 72 h.
b, expression of HLA class I heavy chain (MHC I),
2M, TAP-1, TAP-2,
LMP-2, and LMP-7 in each cell line with or
without IFN-
induction (1000 units/ml for 72 h). Total RNA
loading to each well was shown as 28 S rRNA (28S) and 18 S
rRNA (18S). c, transfection with wild type
TAP-1, but not vector alone, restored HLA class I expression
in the SK-MEL-19 cells. SK-MEL-19 cells were transfected with either
vector alone (top panels) or vector with TAP-1
cDNA insert (bottom panels). These stable clones from
each group were stimulated with or without IFN-
and analyzed for
cell surface HLA-A, -B, and -C, as detailed in a.
2M as well as HLA class I heavy chain, a Northern blot
analysis was performed to detect the expression of these genes (Fig.
1b). In 1102 and 1195 cells, all six genes were expressed at
low but detectable levels. IFN-
treatment drastically induced expression of all six genes. Interestingly, in the SK-MEL-19 cells, whereas
2M, HLA heavy chain, LMP-2,
LMP-7, and TAP-2 were present at low levels
without induction, no TAP-1 mRNA was detected. After IFN-
treatment,
2M, HLA heavy chain,
TAP-2, LMP-2, and LMP-7 were expressed
at high levels, yet TAP-1 was still expressed at low levels.
-treatment. Moreover, the TAP-1 transfectants were as
responsive to IFN-
as the other melanoma cell lines. Based on these
results, it is likely that the primary defect of antigen presentation
in SK-MEL-19 cells is attributable to defects in TAP-1 expression.
T replacement was identified at position
446 (the first ATG of the TAP-1 gene is designated as +1),
which is close to the first transcription start site at
427 (20)
(Fig. 2a). Because the T
allele results in a loss of restriction site AflIII, we did
a Southern blot hybridization using AflIII to confirm the
mismatch. As shown in Fig. 2a, whereas the HeLa cell line
contained homozygous G alleles as described previously (20), both
SK-MEL-19 and the breast cancer cell line SK-BR-3 were homozygous for T
alleles that lack the restriction site for AflIII. To
test whether this single-nucleotide replacement results in reduced
promoter activity, both alleles of the TAP-1 promoter were
cloned into the pGL2-basic vector that contains the luciferase reporter
gene. As shown in Fig. 2b, the T allele TAP-1
promoter retained 50% of the promoter activity compared with the G
allele. However, given the significant variation in transient
transfection and luciferase assays, it is unclear whether the G
T
change has a significant effect on TAP-1 transcription. However, both promoters were equally efficiently induced by IFN-
treatment, whereas the TAP-1 mRNA in the original
SK-MEL-19 cell line was not induced by IFN-
treatment (Fig.
1b). Moreover, our analysis of normal human peripheral blood
lymphocyte samples revealed that both alleles were present at a high
frequency, and individuals that carry either G or T alleles have
equivalent cell surface HLA class I antigen expression (data not
shown).
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Fig. 2.
Posttranscriptional mechanisms are
responsible for poor accumulation of TAP-1
mRNA. a, a single-nucleotide polymorphism,
adjacent to the first transcription start site ( 427), was identified
at
446 in the bidirectional promoter shared by the TAP-1
and LMP-2 genes. The G
T change results in the loss of
the AflIII restriction site. Southern blot hybridization was
performed using AflIII and detected by a DNA probe that
encompasses the downstream region of the polymorphism site. SK-MEL-19
cells showed one 5.6-kb band that represents homozygous T allele, as
did the breast cancer cell line SK-BR-3, which has significant cell
surface HLA class I surface expression (data not shown). HeLa cells, in
contrast, are homozygous for the G allele. b, activities of
T and G alleles of TAP-1 promoter (pTAP1/T and
pTAP1/G, respectively) in SK-MEL-19 cells. The
two allelic forms of TAP-1 promoter were cloned into
pGL2-basic vector (basic) that did not contain any promoter
or enhancer but encoded firefly luciferase. The pGL2-SV40 construct
(SV40) that had both SV40 promoter and SV40 enhancer as well
as the firefly luciferase reporter gene was used as positive control.
After transfection, IFN-
was added to the cell culture at 1000 units/ml. Cells were lysed 48 h after transfection, and luciferase
expression was tested using a luminometer. Data shown are
representative of at least five independent experiments. c,
the TAP-1 gene was actively transcribed in SK-MEL-19 cells
in the presence and absence of IFN-
in nuclear run-on assay.
Endogenous GAPDH expression was used as a positive control,
and the pcDNA3.1/Hyg(+) vector was used as a negative control. The
run-on experiments were repeated three times with similar
results.
appeared to up-regulate
TAP-1 transcription somewhat. In contrast, LMP-2
was transcribed at an undetectable level but was induced to high levels
by IFN-
(Fig. 2c). The lack of LMP-2
transcription at basal condition may reflect the IFN-
-inducible
expression pattern of this gene. These results demonstrate that lack of
TAP-1 mRNA in SK-MEL-19 cells was not due to defective
transcription. Taken together, the results demonstrate that a
posttranscriptional defect is responsible for poor TAP-1
expression in SK-MEL-19 cells even after IFN-
stimulation. Numerous
studies have revealed defective TAP-1 expression among tumor
cells (7, 23). To our knowledge, however, this is the first example of
a posttranscriptional defect of TAP-1 expression.
(1000 units/ml) for 48 h at 37 °C. At different
time points after the CHX was added to the cell culture, cells were
harvested, and the total cellular RNA was analyzed for TAP-1
mRNA. The intensity of each band was quantified using ImageQuant
5.0 software (Amersham Biosciences) after exposure to a
phosphorimaging screen (Fig. 3). For
a better comparison, TAP-1 mRNA levels were
normalized to the endogenous housekeeping gene GAPDH level,
and the fold increase compared with non-CHX-treated cells was
calculated. Under basal conditions, the TAP-1 mRNA was
up-regulated 4.8-fold in SK-MEL-19 cells. After IFN-
induction, the
TAP-1 mRNA was up-regulated 25.7-fold. In comparison,
CHX caused a less significant increase of TAP-1 mRNA in
both IFN-
-treated and untreated HeLa cells. Taken together, the lack
of TAP-1 mRNA, the normal transcription of
TAP-1, and the rescue of TAP-1 mRNA by CHX
treatment suggest that the TAP-1 mRNA was rapidly
degraded in the SK-MEL-19 cells. It is noteworthy that in SK-MEL-19
cells, the effect of CHX was significantly stronger when used in
combination with IFN-
. This finding cannot be fully explained by the
fact the IFN-
is a transcriptional activator for antigen
presentation genes, because its effect on TAP-1
transcription is not so obvious in SK-MEL-19 cells as shown in Fig.
2c. It is likely that IFN-
stabilized mRNA in
SK-MEL-19 cells, although this possibility remains to be tested
formally.
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Fig. 3.
TAP-1 mRNA level in SK-MEL-19
cells was increased by CHX. The protein synthesis inhibitor CHX
was added to the SK-MEL-19 cells and HeLa cells that had normal
TAP-1 and HLA class I expression. Total RNA was isolated
from both cells at different time points and subjected to Northern blot
hybridization to detect TAP-1 expression. The blot was
exposed to a PhosphorImager, and the signal intensity was quantified
using ImageQuant 5.0 software (Amersham Biosciences). After
normalization of TAP-1 signal to endogenous GAPDH
signal in each sample, the signals in the CHX-treated group were
compared with those that received no CHX treatment. These signals were
quantitated as fold of those in untreated cells.
and CHX as
described above. All of the three clones sequenced showed a single-nucleotide deletion at position +1489 (Fig.
4a), which resides in exon 7 in the TAP-1 gene. Further analysis showed that multiple
downstream premature termination codons (the closest one is at position
+1555) were present due to this nucleotide deletion (two of them are
shown in Fig. 4c). To confirm that the mutation was in the
TAP-1 gene, we amplified exon 7 of the TAP-1 gene
from the SK-MEL-19 cells by PCR. The PCR products were digested with
BslI because this restriction enzyme recognizes the deletion mutant but does not recognize the wild type exon 7. Because complete digestion was obtained, it appears that the SK-MEL-19 cells are homozygous for the frameshift mutation (Fig. 4b), even
though the cytogenetic analysis revealed that there are four copies of chromosome 6 present in the SK-MEL-19 cells (data not shown). We
subsequently amplified exon 7 of the TAP-1 gene from 50 normal human peripheral lymphocyte genomic DNA samples by PCR and
subjected the PCR products to BstI digestion. Because none
of the PCR products from the 50 samples could be digested by
BslI, it is most likely that the single-nucleotide deletion
in SK-MEL-19 cells resulted from a somatic mutation (data not shown)
and that the apparent homozygosity of the TAP-1 locus caused
aneuploidy.
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Fig. 4.
A homozygous single-nucleotide deletion was
identified in the TAP-1 gene at position +1489 that
resulted in premature termination codons. a, sequencing
chromogram. The arrow points to the deletion site.
b, primers hTAP1E7.f and hTAP1E7.r (arrows) were
used to amplify the deletion region in TAP-1 exon 7 (E7). The arrowhead points to the position of the
deletion resulting in a new BslI site. The PCR products of
genomic DNA were purified and digested with BslI. Gel
electrophoresis data showed that SK-MEL-19 cells were homozygous for
the +1489 deletion. U, uncut; B,
BslI-digested; M, molecular weight. c,
the sequence of the deletion region is shown, and the downstream
premature termination codons (X) are
underlined.
View larger version (59K):
[in a new window]
Fig. 5.
The single-nucleotide deletion D1489 in the
TAP-1 gene results in accelerated decay of the TAP-1
mRNA by a non-NMD-related mechanism. RNase protection assay of
three Tet-Off SK-MEL-19 cell clones stably transfected with TAP-1 WT,
TAP-1 D1489, and TAP-1 Del3 cDNA constructs was conducted using the
antisense transcripts as probe. a, depiction of the Tet-Off
system and the pBI-EGFP/TAP1 constructs containing TAP-1 WT, TAP-1
D1489, and TAP-1 Del3 used to measure the mRNA half-lives. The
nucleotide sequences around the mutation site in the cDNA
constructs are denoted. b, decay kinetics of the TAP-1 WT,
TAP-1 D1489, and TAP-1 Del3 mRNA. Means ± S.D. of four
experiments are presented. The half-lives of the mRNA were
determined by manual best-fit regression analysis. c, RNase
protection assay to demonstrate the accelerated decay of TAP-1 D1489
and TAP-1 Del3 message compared with that of the wild type TAP-1. The
antisense probe protects a 345-nucleotide TAP-1 mRNA.
One representative assay for each TAP-1 construct is
shown.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jian-Xin Gao for flow cytometry and Jennifer Kiel and Lynde Shaw for secretarial assistance.
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FOOTNOTES |
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* This work was supported by NIH Grants CA82355, CA69091, and CA58033; Department of Defense Grant DAMD17-00-1-0041; and The Ohio State University Comprehensive Cancer Center.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.
¶ To whom correspondence should be addressed: Dept. of Pathology and Comprehensive Cancer Center, The Ohio State University Medical Center, 129 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-292-2003; Fax: 614-688-8152; E-mail: zheng-1@medctr.osu.edu.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M300954200
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ABBREVIATIONS |
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The abbreviations used are:
TAP, transporter
associated with antigen processing;
LMP, low molecular weight peptide;
NMD, nonsense-mediated mRNA decay;
CHX, cycloheximide;
Tet, tetracycline;
2M,
2-microglobulin;
PE, phycoerythrin;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
EGFP, enhanced green fluorescent protein;
WT, wild type.
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