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First Department of Internal Medicine and Department of Neurosurgery (H.K., S.I., M.T., T.S.), Gunma University School of Medicine, Maebashi 371-8511, Japan
Address all correspondence and requests for reprints to: Masami Murakami, M.D., First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan. E-mail: mmurakam{at}showa.gunma-u.ac.jp
Abstract
Type II iodothyronine deiodinase (DII) messenger ribonucleic acid (mRNA) and its activity have been demonstrated in human normal brain. Although DII activity has been demonstrated in brain tumors, expression of DII mRNA has not been studied in these tumors. To investigate the mechanisms involved in the expression of DII activity in brain tumors, we studied DII mRNA and DII activity in astrocytoma (two cases), glioblastoma (three cases), and oligodendroglioma (one case). DII mRNA, the size of which was indistinguishable from that in control cerebral cortical tissue, was demonstrated in all of the brain tumors tested, although the intensity of the hybridization signal showed wide variation among the tumors. DII activity was also detected in all tumors. DII mRNA and DII activity were highest in the tissue from oligodendroglioma. A significantly positive correlation was observed between DII mRNA and DII activity in these tumors (r = 0.94; P < 0.01), suggesting that DII expression in brain tumors is regulated at the pretranslational level. The present results demonstrate, for the first time, that DII mRNA as well as DII activity are expressed in brain tumors, and that DII mRNA is significantly correlated with DII activity in those tissues.
THYROID HORMONES play important roles in normal brain maturation and normal brain function (1, 2). It has also been suggested that thyroid hormones may have pathophysiological roles in the development of brain tumors. Thyroid hormone receptors have been identified in brain tumors (3), and thyroid hormones are suggested to be involved in the proliferation of brain tumor cells (4).
T4, which is a major secretory product of the thyroid gland, needs to be converted to T3 by iodothyronine deiodinase to exert its biological activity (5). Two different isozymes have been demonstrated for the iodothyronine deiodinase to catalyze T4 activation (5). Type I iodothyronine deiodinase (DI) is present in thyroid gland, liver, kidney, and many other tissues, whereas type II iodothyronine deiodinase (DII) is present in a limited number of tissues, including brain, anterior pituitary, brown fat, and pineal gland, in the rat (5). DI activity is known to decrease in the hypothyroid state and is believed to have a primary role in maintaining circulating T3 levels (5). DII activity, in contrast, increases in the hypothyroid state and plays a critical role in providing local intracellular T3 (5). DII activity has been demonstrated in cultured fetal rat brain cells (6), cultured rat glial cells (7, 8), and mouse neuroblastoma cell line (9). In humans, DII activity has been demonstrated in normal brain tissues and brain tumors (10, 11).
Recently, a complementary DNA (cDNA) encoding DII was cloned from Rana catesbeiana tissues (12), and its mammalian counterpart was subsequently isolated from rat brown fat (13). DII messenger ribonucleic acid (mRNA) has been demonstrated in rat brain (13), cultured rat astrocytes (14), and normal human brain (13). However, it is suggested that the mammalian homologue of frog DII does not encode a functional DII enzyme in rat astrocytes (15), and DII mRNA in brain tumor tissues has not been demonstrated. It is, therefore, of importance to study whether DII mRNA is expressed in brain tumor tissues and whether its mRNA levels correlate with functional DII enzyme.
In the present report Northern analysis of DII mRNA as well as measurement of DII activity in brain tumor tissues were performed to investigate the correlation between DII mRNA and DII activity in brain tumors.
Subjects and Methods
Subjects and tissue preparation
At the time of surgery, brain tumor tissues were obtained from
Japanese patients with astrocytoma (cases 1 and 2), glioblastoma (cases
3- 5), or oligodendroglioma (case 6), who did not have thyroid
diseases. Clinical findings are summarized in Table 1
. Steroid treatment was performed in
cases 2 and 5 before and/or during surgery, and radiation therapy was
performed in case 2 before surgery. Control cerebral cortical tissue
was obtained from an autopsy case (58-yr-old Japanese female). Each
tissue was snap-frozen in liquid nitrogen and stored at -70 C until
determination of DII mRNA and DII activity. Informed consent was
obtained from the patients for the use of brain tumor tissues. The use
of the tissues did not adversely affect the clinical diagnosis or
treatment of the patients.
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[
-32P]UTP and
[125I]T4 were purchased
from NEN Life Science Products (Boston, MA). LH-20 was
obtained from Pharmacia (Uppsala, Sweden). AG 50W-X2 resin and protein
assay kit were obtained from Bio-Rad Laboratories, Inc.
(Hercules, CA). T7 and SP6 RNA polymerase were purchased from Nippon
Gene (Tokyo, Japan). All other chemicals at the highest quality were
obtained from Sigma (St. Louis, MO) or Wako Pure Chemical Industries, Ltd.. (Osaka, Japan) unless otherwise
indicated.
RNA isolation and Northern analysis
Total RNA was isolated from brain tumor tissues and control
cerebral cortical tissue by modified acid guanidinium thiocyanate
phenol-chloroform method according to the method of Chomczynski and
Sacchi (16). Northern analyses were performed as
previously described (17). Human DII cDNA fragment
containing residues 110-1051 (numbering of residues as GenBank
accession no. U53506) was synthesized by RT-PCR from total RNA isolated
from the thyroid tissue of a patient with Graves’ disease
(17). Human glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) cDNA containing residues 71–1053 was also cloned as described
previously (17). Human DII cDNA and human G3PDH cDNA
cloned into pCRII (Invitrogen, San Diego, CA) were used to
synthesize complementary RNA (cRNA) probe. The cRNA probes for human
DII and human G3PDH were synthesized with
[-32P]UTP and SP6 RNA polymerase or T7 RNA
polymerase, respectively. Ten micrograms of total RNA per lane were
electrophoresed on a 1.4% agarose gel containing 2 mol/L formaldehyde
and transferred overnight in 20 x SSC (1 x SSC = 150
mmol/L sodium chloride and 15 mmol/L trisodium citrate) to a nylon
membrane (Biodyne, Pall BioSupport Corp., East Hills, NY). RNA was
cross-linked to the nylon membrane with a UV Stratalinker. The
membrane was prehybridized with the hybridization buffer (50%
formamide, 0.2% SDS, 5% dextran sulfate, 50 mmol/L HEPES, 5 x
SSC, 5 x Denhardt’s solution, and 250 µg/mL denatured salmon
sperm DNA) at 68 C for 2 h. Subsequently, the membrane was
hybridized at 68 C overnight with the hybridization buffer containing a
human DII cRNA probe. The membrane was washed twice in 2 x
SSC-0.1% SDS at 25 C for 15 min and twice in 0.1 x SSC-0.1% SDS
at 68 C for 1 h. Autoradiography was established by exposing the
filters for 3–12 h to x-ray film (Kodak XAR-2,
Eastman Kodak Co., Rochester, NY) at -70 C. After
detection of DII mRNA, the probe was stripped off, and blots were
rehybridized with human G3PDH cRNA probe as a control. Hybridization
and washing were performed as described above, and the membrane was
exposed for 1 h. mRNA levels were quantitated by densitometry
using NIH Image (version 1.61), and the optical density of the DII band
of 7.5 kb in length was corrected for G3PDH. RNA samples for comparison
were analyzed on the same blot.
Measurement of DII activity
DII activity was measured as previously described
(18) with minor modifications (19). Brain
tumor tissues and control cerebral cortical tissue were homogenized
separately in a 10-fold volume of assay buffer (100 mmol/L potassium
phosphate, pH 7.0, containing 1 mmol/L ethylenediamine tetraacetate and
20 mmol/L dithiothreitol). After centrifugation at 3000 rpm for 15 min,
the supernatants were incubated with 2 nmol/L
[125I]T4, which was
purified using LH-20 column chromatography on the day of the
experiment, in the presence of 1 mmol/L 6-propyl-2-thiouracil for
1 h at 37 C in duplicate. The reaction was terminated by adding
100 µL 2% BSA and 800 µL 10% trichloroacetic acid. After
centrifuging at 3000 rpm for 10 min, the supernatant was applied to a
small column packed with AG 50W-X2 resin (bed volume, 1 mL) and eluted
with 2 mL 10% glacial acetic acid. Separated
125I was counted with a 
-counter. Nonenzymatic
deiodination was corrected by subtracting
I- released in tissue-free tubes. The
protein concentration was determined by Bradford’s method using BSA as
a standard (20). The deiodinating activity was calculated
as femtomoles of I- released per mg protein/h
after multiplication by a factor of 2 to correct random labeling at the
equivalent 3'- and 5'-positions.
Statistics
First order regression analysis of correlation between DII activity and DII mRNA in brain tumor tissues was performed.
Results
Northern analysis of DII mRNA in brain tumors
Figure 1 demonstrates Northern
analysis of DII mRNA using human DII cRNA probe and human G3PDH cRNA
probe in control cerebral cortical tissue, astrocytoma (cases 1 and 2),
glioblastoma (cases 3–5), and oligodendroglioma (case 6). The
hybridization signal of DII mRNA (
7.5 kb) was clearly demonstrated
in control cerebral cortical tissue. DII mRNA of identical size was
demonstrated in all of the brain tumors tested, although the intensity
of the hybridization signal varied among the tumors.
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DII activity was measured in brain tumor tissues. DII mRNA was
quantitated by densitometry and corrected for G3PDH mRNA. Figure 2 shows the DII mRNA levels and DII
activities in these tumor tissues. DII activity was detected in all of
the tumors. Both DII mRNA and DII activity were highest in the tissue
from anaplastic oligodendroglioma (case 6). The relatively low levels
of DII mRNA and DII activity found in case 2 with astrocytoma might be
due to the previous radiation therapy (Table 1
).
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demonstrates a correlation
between DII mRNA and DII activity in brain tumors. A significant
positive correlation was observed between DII mRNA and DII activity in
these tumors (r = 0.94; P < 0.01).
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The present results clearly demonstrated that DII mRNA, the size of which was indistinguishable from that in control cerebral cortical tissue, and DII activity were present in all of the brain tumor tissues tested, including astrocytoma, glioblastoma, and oligodendroglioma. Among the tumors examined, both DII mRNA and DII activity were highest in the tissue from anaplastic oligodendroglioma. DII mRNA showed a significant positive correlation with DII activity in brain tumor tissues in the present study.
Although DII mRNA has been demonstrated in rat brain (13), cultured rat astrocytes (14), and normal human brain (13), DII mRNA has not been demonstrated in human brain tumors, and it is not known whether DII mRNA correlates with DII activity in human brain. The present study demonstrated, for the first time, the presence of DII mRNA in brain tumors. Both DII mRNA and DII activity showed wide variations among the tumors in the present study. One of the reasons for the difference in DII expression in brain tumors in the present study may be the difference in treatment before and/or during surgery. Previous radiation therapy might have decreased DII expression in case 2 with astrocytoma. As glucocorticoid has been demonstrated to increase DII activity in cultured rat astroglial cells (21), steroid treatment might have altered DII expression in brain tumors, such as case 5 with glioblastoma. In any case, it is noteworthy that a significant positive correlation is observed between DII mRNA and DII activity in brain tumors in the present study. DII is demonstrated to be highly expressed in human astroglial cell tumors in the present study, which is in agreement with the recent observations indicating that DII is primarily expressed in astroglial cells in the central nervous system in the rat (22, 23).
Nucleotide sequence analysis of cloned DII cDNA has revealed that DII contains unique in-frame TGA codons that code for selenocysteine (13). A stem-loop selenocysteine insertion sequence (SECIS element) is required for the read-through and translation of the TGA codons into selenocysteine. However, the classical SECIS element is not identified in the reported partial rat DII cDNA clone, and its partial cDNA is able to express deiodinase activity when fused to the SECIS containing the 3'-untranslated region of the rat type III iodothyronine deiodinase cDNA (13). Recently, a functional SECIS element was identified several kilobases downstream of the TGA codon in the 3'-untranslated region of mouse (24) and human (25) DII genes. Although DII mRNA has been demonstrated in rat astrocytes, and DII activity in rat astrocytes has been shown to be selenium dependent (14), it is suggested that the cloned mammalian homolog of frog DII cDNA does not encode a functional DII enzyme in rat astrocytes (15). Therefore, it is controversial whether the reported cDNA for DII encodes functional DII enzyme in glial cells. The significantly positive correlation between DII mRNA, measured using the cloned human DII cRNA probe, and DII activity observed in brain tumors in the present study suggests that cloned human DII cDNA encodes a functional DII enzyme in human glial cells. The present results also suggests that DII expression in brain tumors is regulated at the pretranslational level.
Several lines of evidence suggest that thyroid hormones are involved in tumorigenesis and tumor growth. It has been demonstrated that thyroid hormones are required for malignant transformation of cultured cells by ionizing irradiation or chemical induction (26, 27). Furthermore, thyroid hormone receptor interacts with the tumor suppressor gene p53, resulting in a modulation of its own transcriptional activity (28). In clinical studies, it has been reported that hypothyroidism prolongs the life span of patients with a variety of cancers (29). In central nervous system tumors, thyroid hormone receptors have been demonstrated in the tissues of astrocytoma and glioblastoma (3). It has been demonstrated that thyroid hormone depletion inhibits the proliferation of astrocytoma, indicating the role of thyroid hormones in the proliferation of brain tumors (4). Based on these observations, it is suggested that local T3 production by DII in brain tumors may be involved in tumorigenesis and tumor growth.
It is noteworthy that DII is highly expressed in anaplastic oligodendroglioma tissue in the present study. Among the glial cells, it is well known that thyroid hormones play important roles at multiple steps in the development of oligodendrocytes, including the proliferation of the committed preprecursor oligodendrocytes, the regulation of the numbers of oligodendrocytes by directly promoting their differentiation, and the maturation of postmitotic oligodendrocytes by stimulation of the expression of various myelin genes, such as myelin basic protein, proteolipid protein, and myelin-associated glycoprotein (30). Increased T3 production by DII in anaplastic oligodendroglioma may play roles related to these functions of thyroid hormones in oligodendrocytes, which remain to be elucidated in further studies.
In conclusion, the present results demonstrate, for the first time, that DII mRNA is expressed in brain tumors, and DII mRNA is significantly correlated with DII activity in those tumors. Further studies are required to clarify the pathophysiological roles of local T3 production by DII in brain tumors.
Acknowledgments
We are indebted to Drs. Tetsuo Negishi, Makoto Imamura, and Takayuki Ogiwara for useful discussion.
Footnotes
1 This work was supported in part by Grant-in-Aid for Scientific
Research 09671024 (to M.Mu.) from the Ministry of Education, Science,
and Culture, Japan. 
Received March 6, 2000.
Revised July 18, 2000.
Accepted August 2, 2000.
References
This article has been cited by other articles:
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  A. C. Bianco, D. Salvatore, B. Gereben, M. J. Berry, and P. R. Larsen Biochemistry, Cellular and Molecular Biology, and Physiological Roles of the Iodothyronine Selenodeiodinases Endocr. Rev., February 1, 2002; 23(1): 38 - 89. [Abstract] [Full Text] [PDF]
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