Activation of Nicotinamide N-Methyltransferase Gene Promoter by Hepatocyte Nuclear Factor-1ß in Human Papillary Thyroid Cancer Cells
Jimin Xu,
Marco Capezzone,
Xiao Xu and
Jerome M. Hershman
Endocrinology and Diabetes Division, Veterans Affairs Medical Center, University of California at Los Angeles School of Medicine, Los Angeles, California 90073
Address all correspondence and requests for reprints to: Jerome M. Hershman, M.D., Endocrinology Division-111D, Veterans Affairs Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: jhershmn{at}ucla.edu.
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ABSTRACT
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We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 27) and lower (e.g. BHP 149) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 27 and BHP 149 cells. In BHP 27 cells, promoter activity was dependent on an upstream sequence. In BHP 149 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1ß (HNF-1ß) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1ß was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1ß-positive BHP 27 cells, but not in the HNF-1ß-negative BHP 149 cells. HNF-1ß bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1ß-specific antibody. Cotransfection of a HNF-1ß expression plasmid increased NNMT promoter activity significantly in both HNF-1ß-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1ß in BHP 149 cells increased endogenous NNMT protein levels. In summary, HNF-1ß functions as a transcription activator for NNMT gene expression in some papillary thyroid cancer cells.
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INTRODUCTION
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PAPILLARY THYROID CANCER is the most common thyroid cancer, comprising 81% of new cases (1). We previously demonstrated that the nicotinamide N-methytransferase (NNMT) gene was overexpressed in many papillary thyroid cancers and cell lines (2). The expression was low or undetectable in other papillary, follicular and Hurthle cancers and cell lines, anaplastic and medullary cancer cell lines, normal thyroid cells, and benign thyroid conditions.
NNMT is a methytransferase structurally and functionally related to thioether S-methytransferase and phenylethanolamine N-methytransferase (3). NNMT has broad substrate specificity. It catalyzes the N-methylation of nicotinamide, pyridines, and other structural analogs and is involved in the biotransformation of many drugs and xenobiotic compounds (4). Human liver displays a significant variation in NNMT activity among individuals. This variation may contribute to the difference in metabolism, therapeutic effect, and toxicity of drugs (3). Nicotinamide, a favorite substrate of NNMT, is used for the synthesis of nicotinamide adenine dinucleotide (NAD+) via a salvage pathway and is an inhibitor of the DNA repair enzyme poly (ADP-ribose) polymerase (5) and the NAD+-dependent histone/protein deacetylases, such as silent information regulator 2 (Sir2) (yeast) and sirtuin 1 (human) (6, 7, 8). Sirtuin 1 deacetylates p53 tumor suppressor and inhibits the p53-dependent apoptotic pathway (9, 10). In yeast, enzymes involved in a nuclear NAD+ salvage pathway have been reported to have effects on Sir2-dependent gene silencing. Inactivation of nicotinate phosphoribosyltransferase by gene mutation caused defects in Sir2-dependent gene silencing (11). Depletion of nicotinamide by increased expression of nicotinate phosphoribosyltransferase gene or nicotinamidase gene increased the Sir2-dependent silencing (12, 13, 14), whereas exogenous nicotinamide decreased Sir2-dependent gene silencing (6). As NNMT irreversibly converts nicotinamide into N (1)-methylnicotinamide, its potential role in the regulation of the NAD+-dependent histone/protein deacetylase activity is implied. The effect of NNMT on Sir2 activity by modulating nicotinamide level was investigated in yeast. It was shown that manipulating expression of human NNMT or its yeast homolog (YLR285W) had effects similar to those from manipulating nicotinamidase on Sir2-dependent gene silencing and yeast lifespan (14). Recently, NNMT was suggested to be involved in the pathogenesis of Parkinsons disease (15, 16, 17). It is believed that overexpression of NNMT in parkinsonian cerebellum may increase the level of toxic pyridinium ions and decrease NAD+ level, causing a decrease in the viability of dopaminergic neurons over a lifetime (17).
NNMT is highly expressed in the liver and weakly expressed in heart, placenta, lung, skeletal muscle, and kidney (3). In addition to papillary thyroid cancer cells, abnormal expression of NNMT has been reported in many situations (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). The mechanisms for the abnormal expression of NNMT in all situations are unknown. In this work, we studied the molecular mechanism leading to overexpression of NNMT in papillary thyroid cancer cell lines. We found that hepatocyte nuclear factor-1ß (HNF-1ß) is expressed in many papillary cancer cell lines and activates NNMT promoter. HNF-1ß was initially identified as a liver-specific transcription factor (26). It binds to DNA either as a homodimer (HNF-1ß/ß) or heterodimer (HNF-1
/ß) with HNF-1
, a functionally and structurally related protein, to regulate target gene expression (26, 27, 28, 29, 30, 31, 32, 33, 34). HNF-1ß and HNF-1
are highly homologous in three regions, the dimerization domain, B domain, and homeodomain, but differ in the transcription activation domain. HNF-1ß expression has been detected in a variety of tissues, such as liver, kidney, intestine, stomach, and pancreas (35, 36, 37). Mutations in the HNF-1ß gene cause maturity-onset diabetes of the young type 5 (38, 39). In this study, we showed that HNF-1ß is expressed in many papillary thyroid cancer cell lines and is involved in the activation of NNMT transcription.
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RESULTS
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Cloning and Expression of NNMT Promoter in Human Papillary Thyroid Cancer Cells
NNMT promoter sequence from 2607 to + 1 relative to the translation initiation codon was amplified from genomic DNA isolated from papillary thyroid cancer cell BHP 1821, a line that expresses endogenous NNMT at a high level (2). The PCR fragment was digested with restriction enzymes, KpnI and MluI, and cloned into corresponding sites in the luciferase reporter gene vector, pGL3-basic. The sequence of the DNA clone was confirmed to be identical to the NNMT genomic sequences in the GenBank database.
To determine whether the cloned NNMT sequence carries essential elements for expression in papillary thyroid cancer cells, the resulting NNMT luciferase reporter plasmid pKM (Fig. 1
) was transfected into papillary thyroid cancer cell lines with higher NNMT gene expression (BHP 27 and BHP 1821), papillary cancer cell lines with relatively lower NNMT expression (BHP 516, BHP 149, BHP 153, and NPA 87), follicular cancer cell lines (WRO 821, FTC 133, FTC 238, and ML-1A), and liver cancer cell line Hep G2. Results presented in Fig. 1
show that the reporter gene construct was expressed well in all papillary cancer cell lines. The luciferase activity of BHP 27 was more than 500 times that of pGL3-basic vector. In contrast, all the follicular cancer cell lines had very low NNMT promoter activity. This result is consistent with our previous study (2). In addition, NNMT promoter activity in the Hep G2 liver cancer cells was also significantly lower than that in the papillary cancer cells. The cloned NNMT promoter sequence seems to contain important regulatory elements for efficient expression in the papillary thyroid cancer cells.

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Fig. 1. Expression of NNMT Promoter in Different Thyroid Cancer Cell Lines
Schematic representation of NNMT reporter gene constructs is shown in the upper panel. Standard luciferase reporter gene assay was performed as described in Materials and Methods. Cells grown in 12 well-plates were transfected with 0.5 µg of NNMT-firefly luciferase reporter gene construct pKM (lower left panel) or an upstream deletion derivative (pKM 1) (lower right panel) and 25 ng of pRL-CMV (internal control, Renilla luciferase) per well. Relative luciferase activity is shown (firefly luciferase activity/Renilla luciferase activity x 100). Values are the mean ± SD (n = 4 to 6). *, Significant difference (P < 0.01): lower left panel, compared with luciferase activity of follicular cancer cell lines (WRO 821, FTC 133, FTC 238, and ML-1A) or Hep G2 liver cancer cell line; lower right panel, compared with luciferase activity of BHP 149, WRO 821, or Hep G2 cells.
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We previously reported that NNMT mRNA levels in BHP 27 and BHP 1821 cells were significantly higher than that in BHP 516, BHP 149, BHP 153, and NPA 87 cells (2). This difference is not reflected by promoter activity assay with the reporter construct carrying NNMT DNA sequence from 2607 to +1 because this construct expressed well in the two subsets of papillary cancer cells (Fig. 1
). To investigate whether NNMT promoter is regulated by a similar mechanism in the two subsets of papillary cancer cell lines, a 5'-deletion construct (pKM
1) was created by deletion of NNMT upstream DNA sequence between 2706 and 343 from pKM (Fig. 1
). NNMT promoter activity of pKM
1 was measured in two representative papillary cancer cell lines, BHP 27 and BHP 149, as well as in follicular cell line WRO 821 and liver cancer cell line Hep G2. The upstream sequence deletion decreased promoter activity dramatically in BHP 27 cells and moderately in other cell lines. The deletion decreased promoter activity about 15-fold in BHP 27, less than 3-fold in BHP 149, and about 2-fold in WRO 821 and Hep G2 cells (Fig. 1
). These results suggested that the sequence between 2706 and 343 contains important positive regulatory elements for efficient expression of NNMT promoter in BHP 27 cells. The sequence between 343 and +1 is referred to as the NNMT basal promoter region. These results indicated that NNMT promoter might be regulated by different factors in the two different papillary cancer cell lines. In BHP 27 cells, strong NNMT promoter activity was dependent on upstream regulatory elements, whereas in BHP 149 cells, regulatory elements in the basal promoter region alone were able to maintain relatively high promoter activity.
Differential Expression of HNF-1ß in Thyroid Cancer Cells
NNMT is dominantly expressed in the human liver (3). The mechanism for this tissue-specific expression is unknown. Expression of liver-specific transcription factors in the papillary thyroid cancer cells may be responsible for the high level of NNMT expression. The HNF-1 transcription factors, HNF-1
and HNF-1ß, are expressed in liver and other tissues and are responsible for the expression of many tissue-specific genes (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39). Their expression in representative thyroid cancer cell lines and some nonthyroid cancer cell lines was determined by RT-PCR (Fig. 2
). The sizes of the expected PCR products are 234 and 317 bp for HNF-1
and HNF-1ß, respectively. HNF1-ß expression was detected in papillary cancer cell lines, BHP 27, BHP 713, BHP 103, BHP 1821, and TPC 1, liver cancer cell line Hep G2, and prostate cancer cell line LNCaP. The levels of HNF-1ß mRNA in these papillary cancer cells were significantly higher than that in the Hep G2 cells. HNF-1ß expression was not detected in papillary cancer cell lines BHP 516, BHP 149, BHP 153, and NPA 87, follicular cancer cell lines WRO 821, FTC 133, FTC 238, ML-1A, and ML-1B, Hurthle cancer cell line XTC-1, breast cancer cell line MCF-7, and primary thyroid cell cultures, O4 (goiter) and HX5 (normal). HNF-1
mRNA was detected strongly in Hep G2 cells and very weakly in the papillary cancer cell line NPA 87 but not in any other cell lines described above. The mRNA level of HNF-1ß and HNF-1
in Hep G2 cells detected in this study is consistent with the results of a previous study that showed that the transcript level of HNF-1ß was 1020 times lower than that of HNF-1
(27).

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Fig. 2. RT-PCR Analysis for HNF-1ß and HNF-1
RT-PCR analysis for HNF-1ß, HNF-1 , and GAPDH was performed with total RNA as described in Materials and Methods. Thyroid cancer cell lines: papillary, BHP 27, BHP 713, BHP 103, NHP 1821, TPC 1, BHP 516, BHP 149, BHP 153, and NPA 87; follicular, WRO 821, FTC 133, FTC 238, ML-1A and ML-1B; Hurthle, XTC-1. Primary thyroid cell cultures: O4, goiter; HX5, normal. Nonthyroid cancer cell lines: Hep G2, liver cancer; LNCaP, prostate cancer; MCF-7, breast cancer.
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The presence of HNF-1ß protein was analyzed by Western blot with nuclear extracts isolated from representative thyroid cancer cell lines (BHP 27 and BHP 1821, HNF-1ß positive; BHP 149 and WRO 811, HNF-1ß negative) and liver cancer cell line Hep G2 with HNF-1ß specific antibody. As expected, HNF-1ß protein with an expected molecular mass of 61.3 kDa was detected in the nuclear extracts of BHP 27, BHP 1821, and Hep G2 cells and was not detected in the nuclear extracts of BHP 149 and WRO 811 (Fig. 3
). Consistent with RT-PCR analysis (Fig. 2
), the level of HNF-1ß protein in BHP 27 and BHP 1821 cells was much higher than that in Hep G2 cells.

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Fig. 3. Detection of HNF-1ß Protein in Thyroid Cancer Cells by Western Blot
Nuclear proteins (20 µg) were separated on a 10% SDS-polyacrylamide gel. After being transferred to a nitrocellulose membrane, the membrane blot was probed with anti-HNF-1ß antibodies. Cell lines: BHP 27 and BHP 1821, papillary and HNF-1ß positive; BHP 149 (papillary) and WRO 821 (follicular), HNF-1ß negative; Hep G2, liver cancer.
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The expression profiles of HNF-1
, HNF-1ß, and NNMT in the cell lines tested above are summarized in Table 1
. It was found that NNMT gene expression correlated with HNF-1ß gene expression. All papillary thyroid cancer cell lines with high levels of NNMT are HNF-1ß positive, whereas other papillary cancer cell lines, follicular cancer cell lines, and primary normal thyroid cell cultures that are NNMT negative or have relatively lower NNMT gene expression are HNF-1ß negative. These findings suggested that HNF-1ß may be a positive regulator of the NNMT gene in the papillary cancer cells with higher NNMT expression.
NNMT Expression in Nonthyroid Cancer Cells
The three nonthyroid cancer cell lines, Hep G2 (liver cancer), MCF-7 (breast cancer), and LNCaP (prostate cancer), tested above showed different HNF-1ß expression. To determine whether NNMT expression correlates with HNF-1ß expression in these nonthyroid cancer cell lines, NNMT mRNA level was determined by RT-PCR. The results presented in Fig. 4
indicate that NNMT is expressed in the HNF-1ß-positive LNCaP cells, but not in the HNF-1ß-negative MCF-7 cells. These results suggested that HNF-ß might also be required for NNMT expression in some nonthyroid cancer cells. NNMT expression in Hep G2 cells was not detected by RT-PCR under the conditions specified (Fig. 4
). We also tested NNMT, HNF-1
, and HNF-1ß expression in Hep G2 cells from a different source and found that the results (data not shown) were the same as those described in Figs. 2
and 4
. To verify the results of RT-PCR analysis, we performed NNMT catalytic activity assay using cell extracts isolated from the Hep G2 cells. Cell extract isolated from BHP 1821 cells was used as a positive control. Strong NNMT catalytic activity was detected in BHP 1821 cells as reported previously (2), whereas in Hep G2 cells very weak activity was detected (Table 2
). These results are consistent with those of the NNMT mRNA assay, suggesting that poor NNMT expression in the Hep G2 cells is limited at the transcription level.

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Fig. 4. Detection of NNMT Expression in Hep G2, LNCaP, and MCF-7 by RT-PCR
Total RNA was subjected to RT-PCR analysis for NNMT and GAPDH as described in Materials and Methods. RNA isolated from BHP 27 cells was used as a positive control for NNMT.
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Identification of a Functional HNF-1 Binding Site in the NNMT Promoter Region
To determine whether HNF-1ß is directly involved in the regulation of NNMT gene expression, we first screened for HNF-1 binding sites in the cloned NNMT promoter region by DNA sequence analysis. A sequence located between nucleotide 148 and 162 relative to the NNMT translation initiation codon was identified with high homology to the HNF-1 consensus sequence (or idealized binding sequence) (40) (Fig. 5
). To test whether this putative HNF-1 site is functional, TTAA to CATT mutations were introduced into the site in the NNMT luciferase reporter plasmid pKM by site-directed mutagenesis (Fig. 5
). The mutant plasmid, pKMmt, was tested to determine whether the mutation had any effects on NNMT promoter activity in three representative cancer cell lines, BHP 27, Hep G2, and BHP 149, by luciferase reporter gene assay.
In the HNF-1ß-positive BHP 27 cells, the HNF-1 binding site mutation decreased NNMT promoter activity about 5-fold (Fig. 5
). In the Hep G2 cells, which express HNF-1ß at a low level, the HNF-1 binding site mutation also decreased NNMT promoter activity significantly. The effect, however, was significantly smaller than that observed in the BHP 27 cells. This result suggested that the putative HNF-1 site is functional and is important for NNMT promoter activity in the HNF-1ß-positive BHP 27 cells. In contrast, the HNF-1 site mutation had no negative effects on NNMT promoter activity in the HNF-1ß-negative BHP 149 cells. This result was expected because HNF-1ß was not expressed in this cell line.
Results presented in Figs. 1
and 5
indicated that NNMT promoter activity in the HNF-1ß-positive BHP 27 papillary cancer cells is strongly dependent on the HNF-1 site in the basal promoter region and regulatory elements in the upstream sequence. Mutation of the HNF-1 site or deletion of the upstream sequence dramatically decreased promoter activity. Because HNF-1 dependent transcription usually involves cooperation with other transcription factors (34, 41, 42, 43), effects of the NNMT upstream sequence on HNF-1ß function were examined. The NNMT basal promoter constructs with (pKM
1mt) or without (pKM
1) the HNF-1 site mutation were transfected into BHP 27, BHP 149, and Hep G2 cells and promoter activity was measured. The results were similar to those obtained with the constructs carrying the NNMT upstream sequence. In the presence of the HNF-1 site mutation, NNMT basal promoter activity was decreased up to 4- and 2-fold in the HNF-1ß-positive BHP 27 and HepG2 cells, respectively (Fig. 5
). These results suggested that HNF-1ß was able to activate NNMT promoter efficiently in the absence of the upstream regulatory elements. However, possible cooperation between HNF-1ß and a factor binding to the upstream sequence cannot be simply ruled out. In the HNF-1ß-negative BHP 149 cells, the mutation had no significant effects on promoter activity as expected, indicating that NNMT promoter activity in BHP 149 cells is independent of the HNF-1 site. Other unidentified regulatory elements in the basal promoter region may be important for the high NNMT basal promoter activity in BHP 149 cells.
Binding of HNF-1ß to NNMT Basal Promoter Region
To demonstrate that the HNF-1ß protein expressed in the papillary cancer cells binds directly to the HNF-1 site in the NNMT basal promoter region, nuclear extract isolated from BHP 27 cells was first tested for HNF-1ß binding activity by gel retardation assay. The NNMT HNF-1 oligonucleotide was labeled with 32P and used as a DNA probe. A major specific retardation complex was observed (Fig. 6A
). This complex could be competed out by 50-fold excess unlabeled NNMT HNF-1 oligonucleotide and consensus HNF-1 oligonucleotide, but not by the mutant NNMT HNF-1 oligonucleotide. Furthermore, the complex was supershifted by an HNF-1ß-specific antibody (Fig. 6B
) but did not react with an HNF-1
-specific antibody (Fig. 6C
). Because HNF-1
is not expressed in the papillary cancer cells, this complex is assumed to be the homodimer form of HNF-1ß (HNF-ß/ß).
To confirm the results described above, similar experiments were performed with nuclear extract isolated from Hep G2 cells, in which HNF-1
and HNF-1ß expression is well documented. In contrast to that observed with BHP 27 nuclear extract, the major specific protein-DNA complex detected with the Hep G2 nuclear extract was the HNF-1
homodimer complex (HNF-1
/
) (Fig. 6
, A, B, and C). This complex migrates slower than the HNF-1ß/ß complex. It could be competed out by 50-fold excess unlabeled NNMT HNF-1 oligonuclotide or consensus HNF-1 oligonucleotide, but not by the mutant NNMT HNF-1 oligonucleotide. It was supershifted by HNF-1
-specific antibody, but not by the HNF-1ß-specific antibody. The HNF-1ß/ß binding activity was very weak in the Hep G2 cells. Most of the HNF-1ß protein expressed in this cell line was present as a heterodimer with HNF-1
(HNF-1
/ß). The HNF-1
/ß complex is located between the HNF-1
/
and HNF-1ß/ß complexes. This complex was also sensitive to competition by the unlabeled NNMT HNF-1 oligonucleotide and the consensus HNF-1 oligonucleotide, but not the mutant NNMT HNF-1 oligonucleotide. The intensity of this complex was diminished when either HNF-1ß- or HNF-1
-specific antibody was added to the binding reactions. The overall HNF-1ß binding activity, including HNF-1
/ß and HNF-1ß/ß in the Hep G2 cells, was much lower than that in the BHP 27 papillary cancer cells.
In addition to BHP 27, four additional thyroid cancer cell lines were also assayed for HNF-1ß binding activity by gel retardation assay. BHP 27 and Hep G2 nuclear extracts were used as positive controls for HNF-1ß and HNF-1
, respectively. As expected, HNF-1ß binding activity was also detected strongly in nuclear extracts isolated from the HNF-1ß-positive papillary cancer cells, BHP 1821, and BHP 713 (Fig. 7
, lane 3 and 5) but not detected or detected only at very low level in the nuclear extracts of the HNF-1ß-negative cells: BHP 153 (papillary cancer), WRO 821 (follicular cancer), and MCF-7 (breast cancer). HNF-1
/
or HNF-1
/ß binding activity was not detected in any of the cancer cells lines studied except Hep G2. These results are consistent with the assay for HNF-1ß and HNF-1
expression determined by RT-PCR (Fig. 2
and Table 1
).

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Fig. 7. Detection of HNF-1ß Binding Activity in Different Thyroid Cancer Cell Lines by Gel Retardation Assay
Standard gel retardation assay was performed as described in Fig. 6 . Nuclear extracts (NE) isolated from BHP 27 and Hep G2 cells were used as positive controls for HNF-1ß and HNF-1 , respectively. Cell lines: BHP 27, BHP 713, and BHP 1821, papillary and HNF-1ß positive; BHP 153 (papillary) and WRO 821 (follicular), HNF-1ß negative; MCF-7, breast cancer and HNF-1ß negative; Hep G2, liver cancer.
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Transient Expression of HNF-1ß Enhanced NNMT Promoter Activity and Endogenous NNMT Protein Level
The results shown above suggested that HNF-1ß activates NNMT promoter in the HNF-1ß-positive BHP 27 papillary cancer cells by binding to the HNF-1 site in the NNMT promoter region. To further confirm these results, the effect of transient HNF-1ß expression on NNMT promoter activity was tested by cotransfection assay. HNF-1ß cDNA was cloned into the mammalian gene expression vector pcDNA3.1(+) as described in Materials and Methods. The HNF-1ß expression plasmid was cotransfected with the NNMT luciferase reporter plasmid pKM (wild type) or pKMmt (HNF-1 binding site mutant) into Hep G2, BHP 27, BHP 149, or WRO 821 cells. In Hep G2 cells, expression of HNF-1ß increased NNMT promoter activity significantly in a dose-dependent manner (Fig. 8A
). At the highest dose (250 ng/transfection), NNMT promoter activity was increased 2.2-fold. The effect of HNF-1ß on NNMT promoter activity is direct because no activation was observed when the wild-type plasmid pKM was replaced by the HNF-1 site mutant plasmid, pKMmt. In fact, the HNF-1ß-independent promoter activity was inhibited slightly by the HNF-1ß expression plasmid for unknown reasons. In BHP 27 and BHP 149 papillary cancer cells and WRO 821 follicular cancer cells, NNMT promoter activity of the wild-type reporter construct was increased about 3.5-, 2.5-, and 3-fold, respectively, by cotransfection with the HNF-1ß expression plasmid (250 ng/per transfection). Promoter activity of the mutant construct (pKMmt), on the other hand, was inhibited slightly by HNF-1ß overexpression as observed in Hep G2 cells (Fig. 8A
). These results further suggested that HNF-1ß is able to function as a transcription activator of the NNMT promoter in these cells.

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Fig. 8. Transactivation of NNMT Promoter by HNF-1ß
Wild-type (wt) HNF-1 site reporter constructs with (A) or without upstream sequence (B) were cotransfected with HNF-1ß expression plasmid (HNF-1ß) into cells as indicated. Reporter constructs carrying the HNF-1 site mutation (mt) were used as controls. pcDNA3 cloning vector was added to transfections to keep the amount of total plasmid DNA constant if necessary. Transfection efficiency was monitored by cotransfection with 25 ng of pRL-CMV (Renilla luciferase) as described in Materials and Methods. For each cell line, relative luciferase activity of transfection reactions without the HNF-1ß expression plasmid is set at 100%. Values are the mean ± SD (n = 4). *, Significant difference (P < 0.01) compared with cells cotransfected with the pcDNA3.1(+) vector plasmid.
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Similar experiments were also performed with the NNMT basal promoter constructs with BHP 27 and BHP 149 cells. Cotransfection of the HNF-1ß expression plasmid with the wild-type construct, pKM
1, increased NNMT basal promoter activity about 8-fold in BHP 27 cells and 2.5-fold in BHP 149 cells (Fig. 8B
). Under the same conditions, the HNF-1ß expression plasmid had no significant effects on promoter activity of the HNF-1 binding site mutant construct, pKM
1mt, in both cell lines. This result is consistent with the results presented in Fig. 5
and further suggests that the HNF-1ß is able to activate NNMT promoter in the absence of the NNMT upstream sequence. Interestingly, the impact of HNF-1ß on NNMT promoter activity is more notable in BHP 27 cells than in BHP 149 cells.
The effect of HNF-1ß on endogenous NNMT expression was determined in BHP 149 cells that are HNF-1ß negative and have lower NNMT expression (Table 1
). The cells were transfected with the HNF-1ß expression plasmid, pcDNA3.1 vector, or NNMT expression plasmid. Nuclear extracts and cytoplasmic fractions were prepared 48 h after transfection and assayed for HNF-1ß protein and NNMT protein, respectively, by Western blotting with HNF-1ß- and NNMT-specific antibodies. HNF-1ß was detected in the cells transfected with the HNF-1ß expression plasmid but not in the cells transfected with the cloning vector or NNMT expression plasmid (Fig. 9
, upper panel). Accompanying HNF-1ß expression, the endogenous NNMT protein level in BHP 149 cells was significantly enhanced when it was compared with cells transfected with the vector only (Fig. 9
, lower panel). In the cells transfected with the NNMT expression plasmid (positive control), NNMT protein level was also increased as expected. The apparent molecular mass of NNMT protein expressed in the thyroid cancer cells is similar to that identified in the liver cells (29 kDa) (3).

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Fig. 9. Transient Expression of HNF-1ß Increased Endogenous NNMT Protein Level
BHP 149 cells grown in 12-well plates were transiently transfected with 0.5 µg of HNF-1ß expression plasmid, pcDNA vector, or NNMT expression plasmid. Nuclear and cytoplasmic proteins were isolated 48 h after transfection and analyzed for HNF-1ß (upper panel) and NNMT (lower panel) by Western blotting using anti-HNF-1ß and anti-NNMT antibodies. SDS-polyacrylamide gel concentrations are 10% for HNF-1ß and 12.5% for NNMT. An unknown 50-kDa protein was detected by the NNMT antibody across all the lanes with similar intensity. It was used as internal control for sample loading.
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In summary, transient expression of HNF-1ß enhanced NNMT promoter activity significantly in the HNF-1ß-positive BHP 27 and Hep G2 cells and HNF-1ß-negative BHP 149 and WRO 821 cells, and enhanced endogenous NNMT protein level in BHP 149 cells. These results suggest that HNF-1ß functions as a transcription activator of NNMT gene in the papillary cancer cells with strong NNMT expression.
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DISCUSSION
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NNMT was expressed highly in many human papillary thyroid cancers and cell lines (2). The mechanism leading to this overexpression was studied in representative papillary cancer cell lines. Several lines of evidence suggested that HNF-1ß is directly involved in the activation of NNMT gene expression in the papillary cancer cells with strong NNMT expression. In these cells, the expression of HNF-1ß gene was demonstrated by RT-PCR analysis (Fig. 2
), and HNF-1ß protein and DNA binding activity were detected by Western blotting and gel retardation assay (Figs. 3
, 6
, and 7
). A putative HNF-1 binding site was identified in the cloned NNMT promoter region. HNF-1ß bound to this site specifically as a homodimer. Mutations in this site abolished HNF-1ß binding (Fig. 6
) and decreased NNMT promoter activity significantly in the HNF-1ß-positive papillary cancer cells (BHP 27) but not promoter activity in the HNF-1ß-negative papillary cancer cells (BHP 149) (Fig. 5
). Overexpression of HNF-1ß enhanced NNMT promoter activity of the wild-type reporter gene construct significantly in both HNF-1ß-positive and -negative cells but not promoter activity of the construct with the HNF-1 binding site mutation (Fig. 8
). In addition, transient expression of HNF-1ß in BHP 149 cells enhanced the endogenous NNMT protein level (Fig. 9
). The combined results suggest that HNF-1ß is able to function as a transcription activator of NNMT promoter and is at least partially responsible for the high level of NNMT gene expression in the HNF-1ß-positive papillary cancer cells.
In other papillary cancer cells, follicular cancer cells, and primary thyroid cultures that have relatively lower or no NNMT expression, HNF-1ß mRNA was not detected by RT-PCR. HNF-1ß protein or binding activity was not detected or detected only very weakly in representative HNF-1ß-negative cell lines. In addition, mutations in the HNF-1 site had no negative effects on promoter activity in BHP 149 cells. It is likely that the strong NNMT basal promoter activity in the HNF-1ß-negative BHP 149 cells is dependent on some other factors. Because transient expression of HNF-1ß was able to enhance NNMT promoter activity specifically in BHP 149 papillary cancer cells and WRO 821 follicular cancer cells and endogenous NNMT protein level in the BHP 149 cells, the absence of HNF-1ß may be partially responsible for the lower or poor NNMT gene expression in these cells.
The NNMT promoter construct carrying NNMT sequence from 2607 to +1 showed differential expression in papillary cancer cell lines and follicular cancer cell lines. It expressed well in all the papillary cancer cell lines, including those with lower NNMT gene expression, but poorly in the follicular cancer cell lines (Fig. 1
). These results are consistent with our previous data showing that the NNMT gene was expressed poorly in many follicular cancers or cell lines (2). The cloned NNMT promoter may contain essential elements for efficient expression in papillary cancer cells. The construct, however, was unable to reflect the difference in NNMT gene expression between papillary cancer cell lines with high level NNMT gene expression (e.g. BHP 27) and papillary cancer cell lines with relatively lower NNMT expression (e.g. BHP 149). Further study with a 5'-deletion construct in two representative papillary cancer cell lines, BHP 27 and BHP 149, suggested that NMT promoter might be regulated differently in the two cell lines. In the BHP 27 cells, strong NNMT promoter activity was dependent on upstream regulatory elements and a HNF-1 site in the basal promoter region. Deletion of the upstream sequence or mutation of the HNF-1 site dramatically reduced NNMT promoter activity. In BHP 149 cells, the basal promoter region alone was able to maintain relatively high promoter activity, and the HNF-1 site was not functional because of the absence of HNF-1ß expression in this cell line. The upstream sequence was required for full promoter activity in the BHP 149 cells, but was not as important as it was in the BHP 27 cells. In addition, we found that the NNMT basal promoter contained in the 5'-deletion construct, pKM
1, responded strongly to glucocorticoid (dexamethasone) in BHP 27 cells, but insignificantly in BHP 149 cells (Xu, J., and J. M. Hershman, unpublished data), further suggesting that different factors are involved in the regulation of NNMT promoter in the two different cell lines. Because papillary cancers can be caused by different mutations, such as RET and NTRK1 rearrangements and BRAF mutations, the regulation of NNMT by different mechanisms in different papillary cancer cells is possible. By DNA microarray analysis, Frattini et al. (44) reported that papillary thyroid cancers with RET and NTRK1 rearrangements and BRAF mutations had similar but distinct gene expression patterns. We have noticed that the papillary cancer cell lines with high NNMT expression (such as BHP 27 and BHP 1821) are RET/PTC1 rearrangement positive and the papillary cancer cell lines with lower NNMT expression (such as BHP 516, BHP 149, BHP 153, and NPA 87) are RET/PTC1 negative (2). NPA 87 was reported to carry a BRAF mutation (45). The mutations leading to papillary cancer in BHP 516, BHP 149, and BHP 153 cells are unclear. Further work is needed to fully understand the difference in NNMT gene regulation between the two subsets of papillary cancer cell lines. It is possible that additional regulatory elements outside of the cloned NNMT promoter region between 2607 and +1 may be required to mimic endogenous NNMT gene expression in the two subsets of papillary cancer cell lines. These elements may provide additional activation of the NNMT promoter in the cells with high NNMT gene expression (e.g. BHP 27) or repression of NNMT promoter in the cells with relatively lower NNMT expression (e.g. BHP 149). Other factors, such as RNA stability and posttranscriptional regulation, may also affect the steady-state NNMT mRNA level or protein level. All these issues remain to be clarified.
The relationship between HNF-1ß and NNMT expression in the Hep G2 liver cancer cells is complex. We showed that NNMT is not expressed or poorly expressed in this cell line as determined by RT-PCR and catalytic activity assay. HNF-1ß gene expression in Hep G2 cells, as determined by RT-PCR, Western blotting, and gel retardation assay, was much lower than that in the papillary cancer cells that expressed NNMT at high level. The HNF-1ß binding activity in Hep G2 cells was mainly detected as a heterodimer (HNF-1
/ß) with HNF-1
, which was expressed at a much higher level than HNF-1ß. The HNF-1ß/ß binding activity was barely detected. As the HNF-1ß/ß form is highly functional in the papillary cancer cells, the absence of NNMT expression in Hep G2 may be due to insufficiency of the functional HNF-1ß/ß forms. HNF-1
protein is structurally and functionally related to HNF-1ß. It can activate some HNF-1ß-dependent promoters (29, 31). A relatively highly expressed HNF-1
in the Hep G2 cells seems to be unable to compensate for lack of HNF-1ß in the activation of the NNMT gene, assuming that HNF-1ß is important for NNMT expression in the liver cells. Further study is needed to determine the role of HNF-1
in NNMT gene expression in the liver cells. The human
1-antitrypsin gene was activated by HNF-1ß in a cell-dependent manner. It was activated in the human pulmonary epithelial cell line H441 by HNF-1ß, but not in Hep G2 cells (31). This is a situation very similar to the NNMT expression described in this study, implying that other cell-specific factors may be involved in the HNF-1ß dependent activation of NNMT gene. The hypothesis for the requirement of other factors is consistent with the facts that mutations in the HNF-1 site in the NNMT promoter and transient expression of HNF-1ß had significantly less impact on NNMT promoter activity in Hep G2 cells than in BHP 27 papillary cancer cells. These other factors may function cooperatively with HNF-1ß as reported in other systems (34, 41, 42, 43). Alternatively, they may function in an HNF-1ß-independent manner. We have found that the NNMT upstream sequence is very important for NNMT promoter activity in BHP 27 cells, whereas deletion of this sequence had no major effects on the activation of NNMT promoter by HNF-1ß (Figs. 5
and 8
). It is possible that some factors binding to the upstream regulatory elements may function independently of HNF-1ß.
HNF-1ß is a transcription factor normally expressed in tissues such as liver, pancreas, lung, kidney, and intestine. To our knowledge, the expression of HNF-1ß in thyroid cells has not been reported previously. By study of NNMT gene expression, we showed that HNF-1ß is overexpressed in many papillary thyroid cancer cell lines. As HNF-1ß is a transcription factor involved in the expression of genes involved in many different cellular functions, the possible pathological function of HNF-1ß in some papillary thyroid cancers will be a very interesting issue to investigate. It was shown recently that inhibition of HNF-1ß expression caused apoptosis in ovarian clear cancer cells (25). Our future study will focus on the detection of HNF-1ß expression in thyroid cancer specimens and potential implications of HNF-1ß expression on the behavior of the HNF-1ß-positive thyroid cancers.
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MATERIALS AND METHODS
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Cell Culture
Human papillary thyroid cancer cell lines, BHP 27, BHP 1821, BHP 713, BHP 103, BHP516, BHP 149, and BHP 153, were established previously in our laboratory (46). Papillary cancer cell lines NPA 87 and follicular cell line WRO 821 were provided by Dr. Guy Juillard (UCLA, Los Angeles, CA). Papillary cancer cell line TPC 1 was provided by Dr. Sissy M. Jhiang (Ohio State University, Columbus, OH). Follicular thyroid cancer cell lines ML-1A and ML-1B were provided by Dr. Daniela Grimm (University of Regensburg, Berlin, Germany). Follicular cancer cell lines FTC 133 and FTC 238 and Hurthle thyroid cancer cell line XTC-1 were provided by Dr. Orlo Clark (UCSF, San Francisco, CA) and Dr. Peter E. Goretzki (Heinrich Heine University, Dusseldorf, Germany). All cell lines except ML-1A and ML-1B were grown in RPMI 1640 medium [RPMI 1640 medium base (Sigma Chemical Co., St. Louis, MO) supplemented with 2 g/liter sodium bicarbonate, 0.14 mM nonessential amino acids, 1.4 mM sodium pyruvate, and 10% fetal bovine serum, pH 7.2). ML-1A and ML-1B cells were grown in the M199 medium [M199 medium base (Sigma) supplemented with 2.2 g/liter sodium bicarbonate, 10% fetal bovine serum, and 1x PSN antibiotic mixture (Life Technologies, Gaithersburg, MD)]. Human hepatocellular carcinoma cell line Hep G2 (ATCC, Manassas, VA) was grown in Eagles MEM (Sigma) supplemented with 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 10% fetal bovine serum.
Plasmid Construction
To clone the NNMT promoter region, genomic DNA isolated from papillary thyroid cancer cell line BHP 1821 was used as template to amplify NNMT promoter sequence using the Expand High Fidelity PCR System (Roche Clinical Laboratories, Indianapolis, IN). Primers used for PCR amplification were designed based on the NNMT genomic DNA sequences in GenBank (National Center for Biotechnology Information, Bethesda, MD). DNA sequences of the primers are: forward, 5'-ACGGTACCTATTCAGTCATAAAGAAGAATTAGATCCTG-3'; reverse, 5'-TAACGCGTTATGTCTCACTTCTGTACCACTGGA-3'. The sequences underlined at the 5'-ends of the primers were added to create a unique KpnI site at the 5'-end and a unique MluI site at the 3'-end of the PCR fragment. After restriction digestion with KpnI and MluI, the 2.6-kb NNMT promoter DNA from 2607 to +1 relative to the translation initiation codon was cloned into corresponding sites in the firefly luciferase reporter gene vector pGL3-basic (Promega Corp., Madison, WI) to generate NNMT-luciferase reporter plasmid pKM. Plasmid pKM
1 was constructed by deletion of NNMT upstream sequence between 2607 and 343 from pKM. Plasmid pKMmt is a derivative of pKM with mutations in the HNF-1 binding site in the NNMT basal promoter region. The mutations were introduced by a two-step RT-PCR protocol with a mutagenic oligonucleotide (5'-GGTCTATTTCTCTGTTAGTGTCATTCCAACCATCTGTTCTAA-3', nucleotide substitutions underlined) and Pfu DNA polymerase (Stratagene, La Jolla, CA). pKM
1mt was identical to pKM
1 except for mutations in the HNF-1 site.
A HNF-1ß expression plasmid was constructed as follows. Total RNA isolated from BHP 27 cells was reverse transcribed. The reverse transcription products were PCR amplified using Pfu polymerase and HNF-1ß specific primers. The sequences of the primers are: forward, 5'-TTGGATCCTTCCGTCCTTGGAAAATGGTGT-3'; reverse, 5'-GCTCTAGAAGTAAGTGGTGTGTGGGCATCA-3'. The sequences underlined were added to generate unique BamHI and XbaI restriction sites at the 5'- and 3'-ends of the cDNA fragment. The HNF-1ß cDNA was cloned into a mammalian expression vector, pcDNA3.1(+), after being restriction digested with BamHI and XbaI. Similar procedures were also used to construct the NNMT expression plasmid. The sequences of the primers used to clone NNMT cDNA are: forward, 5'-GCGCTAGCCTGAGACTCAGGAAGACAACTTCT-3'; reverse, 5'-TTGGATCCAGGTCAAAGGAATTGCTTTAATTGAG-3'. Unique NheI and BamHI restriction sites (underlined) were added to the forward and reverse primers, respectively. The NNMT cDNA was cloned into corresponding restriction sites in pcDNA3.1(). The expression of both HNF-1ß and NNMT cDNAs was under the control of the cytomegalovirus (CMV) promoter. All plasmid constructs were confirmed by DNA sequencing.
Transfection and Reporter Gene Assay
For transient transfection, about 2 x 105 cells were seeded onto each well of a 12-well plate. After growing about 24 h, the cells were transfected with 0.5 µg of firefly luciferase reporter gene plasmid and 25 ng of Renilla luciferase control reporter gene plasmid pRL-CMV (Promega) with Effectene (QIAGEN, Inc., Valencia, CA). About 18 h after transfection, Effectene-DNA and medium mixture were removed. The transfected cells were grown in fresh growth medium for 28 h. Luciferase activities were then assayed using the dual-luciferase assay kit (Promega). The firefly reporter gene activities were normalized with the control reporter gene activities. Experiments were performed two to three times and data represent mean ± SD.
Semiquantitative RT-PCR
Total RNA was isolated from cultured cells using the RNeasy Mini Kit (QIAGEN). Total RNA (1 µg) was reverse transcribed in a total volume of 10 µl for 90 min at 37 C with the Omniscript RT Kit (QIAGEN) using oligo (dT1113) primers (Life Technologies). The reverse transcription product (1 µl) was subjected to PCR in a total of 50 µl with Taq polymerase. The PCR conditions were: NNMT, 94 C for 30 sec, 56.7 C for 30 sec, 72 C for 30 sec, 26 cycles; HNF-1
, 94 C for 30 sec, 60 C for 30 sec, 72 C for 40 sec, 33 cycles; HNF-1ß, 94 C for 30 sec, 63 C for 30 sec, 72 C for 50 sec, 35 cycles; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 94 C for 30 sec, 57 C for 30 sec, 72 C for 40 sec, 21 cycles. The sequences of the gene-specific primers were: NNMT, 5'-GTTTGGTTCTAGGCACTCTGCA-3' (forward)/5'-GCAGAGAGGAGCTGATAGATAGTGG-3' (reverse); HNF-1
, 5'-AGACACTGAGGCCTCCAGTGAGT-3' (forward)/5'-GAGATGAAGGTCTCGATGACGCT-3' (reverse); HNF-1ß, 5'-AGCTGTCAGGAGTGCGCTACAGC-3 (forward)/5'-GGAGGTGTTGAGGCTTTGTGCA-3' (reverse); GAPDH, 5'-GCTGCCAAGGCTGTGGGCAAGGTC-3' (forward)/5'-TTGTCATACCAGGAAATGAGCTT-3' (reverse). PCR products were separated by agarose gel electrophoresis.
Nuclear Extract Isolation and Gel Retardation Assay
Nuclear extracts were isolated according to the method described previously (47). Double-stranded oligonucleotides carrying a NNMT HNF-1 binding site were end labeled with [
-32P]ATP by polynucleotide kinase. The labeled oligonucleotides were purified with the QIAGEN nucleotide removing kit (QIAGEN) and then used as DNA probe for gel retardation assays. Nuclear extracts (10 µg) were first incubated with 2 µg of nonspecific DNA competitors [poly (DI-dC).(dI-dC)] in a binding buffer [20 mM HEPES, pH 7.8; 5% glycerol, 50 mM KCl; 1 mM EDTA; 1 mM dithiothreitol] for 10 min at room temperature. Specific DNA competitor (when it was used) was added in 50 molar excess to the binding buffer before nuclear extracts were added. About 10 fmol of the DNA probe were added to the binding mixture, and the binding reaction was performed at room temperature for 30 min. Antibodies for HNF-1
(C-19) and HNF-1ß (C-20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies were added 2 µg per reaction 10 min after addition of the DNA probe. The final volume of the binding reaction was 15 µl. DNA-protein complexes were separated by electrophoresis in a 5% polyacrylamide gel in half-strength Tris-borate-EDTA buffer. Radioactive bands were detected by autoradiography. The DNA sequences of the oligonucleotides used as DNA probe or competitors are (top strand only): NNMT HNF-1 binding site, 5'-TATTTCTCTGTTAGTGTTTAACCAACCATCT-3'; NNMT HNF-1 binding site mutant, 5'-TATTTCTCTGTTAGTGTCATTCCAACCATCT-3', mutations underlined; consensus HNF-1 binding site, 5'-TCGATGTGGTTAATGATTAACCGTT-3' (40).
NNMT Catalytic Activity Assay
NNMT catalytic activity assay for cultured cells was performed as described previously (2, 3). Radioactive S-[methyl-14C]adenosyl-1-methionine (SA, 53 mCi/mmol; ICN Biochemicals, Inc., Costa Mesa, CA) was used as the methyl donor and nicotinamide as the methyl acceptor. Radioactivity of the reaction product, N1-[14C]methylnicotinamide, was measured with a liquid scintillation counter (LS6000 SC, Beckman Instruments, Inc., Fullerton, CA) after being separated from the substrates by extraction with isoamyl alcohol/toluene. NNMT activity was expressed as counts per min (cpm) per µg protein.
Western Blotting
Proteins were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to nitrocellulose membranes. The membrane blots were incubated with rabbit polyclonal antibody (1:1000 dilution) against human HNF-1ß (H-85; Santa Cruz Biotechnology) or NNMT (Dr. Richard Weinshilboum, Mayo Clinic, Rochester, MN) for 3 h followed by incubation with horseradish peroxidase-conjugated goat antirabbit IgG (Pierce Chemical Co., Rockford, IL; 1:2000 dilution) for 1.5 h. HNF-1ß and NNMT proteins were visualized using an enhanced chemiluminescent substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Pierce).
Statistical Analysis
Statistical analysis was performed with Students t test with significance at P < 0.05.
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ACKNOWLEDGMENTS
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We thank Dr. Guy Juillard, Dr. Sissy M. Jhiang, Dr. Daniela Grimm, Dr. Orlo Clark, and Dr. Peter Goretzki for providing thyroid cancer cell lines and Dr. Richard Weinshilboum for NNMT antibody. We are grateful to Dr. Gregory A. Brent for helpful suggestions and for his critical review of the manuscript.
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FOOTNOTES
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This work was supported by a grant from Veterans Affairs Medical Research Funds (to J.M.H.).
First Published Online October 14, 2004
Abbreviations: CMV, Cytomegalovirus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HNF-1ß, hepatocyte nuclear factor-1ß; NAD+, nicotinamide adenine dinucleotide; NNMT, nicotinamide N-methytransferase; SDS, sodium dodecyl sulfate; Sir2, silent information regulator 2.
Received for publication May 27, 2004.
Accepted for publication October 5, 2004.
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