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and CYP24) in Human Nonsmall Cell Lung Carcinomas1
Cytochroma, Inc. (G.J., H.R., A.Z., R.C., J.W., M.P.), Bioscience Complex, Kingston, Ontario, Canada K7L3N6; the Departments of Biochemistry (G.J., V.B., M.P.) and Medicine (G.J.); and Pathology and Cancer Research Laboratories (M.P.), Queen’s University, Kingston, Ontario, Canada K7L 3N6
Address all correspondence and requests for reprints to: Dr. Glenville Jones, Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6. E-mail: gj1{at}post.queensu.ca
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Abstract |
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-hydroxylase is
believed to play a major role in the pathogenesis of hypercalcemia
associated with various types of granulomatous and lymphoproliferative
diseases and certain solid tumors. In this paper, we describe the
cloning of the cytochrome P450 component of the extrarenal enzyme from
a human nonsmall cell lung carcinoma, SW 900. The cytochrome P450 for
the extrarenal 1-hydroxylase has an amino acid sequence identical to
that of the cytochrome P450 component of the CYP1, the renal form of
the enzyme, and appears to be a product of the same gene. CYP1
messenger RNA (mRNA) and 1-hydroxylase enzyme activity were detected
in two (SW 900, SK-Luci-6) of a series of five nonsmall cell lung
carcinoma cell lines. All five lung cell lines were cultured with the
same medium under the same conditions, but only two of the five
expressed 1-hydroxylase enzyme; two others (WT-E, Calu-1) expressed
high levels of the reciprocally regulated enzyme, 25-hydroxyvitamin
D3-24-hydroxylase, with its specific cytochrome P450
component, CYP24. Although under basal conditions the lung cell line SW
900 expressed only CYP1 and showed 1-hydroxylase enzyme activity,
when treated with small concentrations of 1,25-dihydroxyvitamin
D3 or high concentrations of 25-hydroxyvitamin
D3, it began to express CYP24 and exhibit 24-hydroxylase
enzyme activity. Somewhat surprisingly, SW 900 cells still had
detectable CYP1 mRNA some 24 h after vitamin D treatment
despite the fact that 1-hydroxylase enzyme activity was
unmeasurable. These data are consistent with the emerging hypothesis
that vitamin D through its active form does not directly turn off
CYP1 mRNA production but, rather, strongly stimulates CYP24, thereby
masking CYP1 activity. The factor(s) responsible for the basal
expression of CYP1 in SW 900 and SK-Luci-6 is currently unknown. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (1-hydroxylase) plays a central role
in calcium homeostasis (1), and elucidation of the details of its
structure and hormonal regulation are expected to provide a wealth of
information (2). The enzyme catalyzes the conversion of
25-hydroxyvitamin D3 (25OHD3) to the hormone
1,25-dihydroxyvitamin D3
[1,25-(OH)2D3], which regulates calcium
and phosphate transport in intestine, bone, and kidney. For at least a
decade after its discovery, it was believed that the 1-hydroxylase
was exclusively expressed in the kidney (3), and this was further
fueled by the knowledge that human patients with chronic renal failure
suffer from avitaminosis D, which results in renal osteodystrophy (4).
Only in the mid-1980s did it become evident that extrarenal cells
(e.g. bone, alveolar macrophage, placenta, and keratinocyte)
could express the 1-hydroxylase enzyme activity in vitro
(5, 6, 7, 8). More recently, Mawer et al. (9), studying a panel of
16 lung cancer cells, showed that one cell line (NCI H82) exhibited
measurable 1-hydroxylase activity when cultured in vitro.
In all reports of extrarenal enzyme activity, the 1-hydroxylase was
not up-regulated by PTH and appeared to be poorly down-regulated, just
the opposite of the renal enzyme, for which tight regulation by plasma
calcium and vitamin D is one of the hallmarks (10). Consequently,
unlike the renal 1-hydroxylase, the extrarenal 1-hydroxylase is
not inversely correlated with the vitamin D-inactivating enzyme
activity, 25-OHD3-24-hydroxylase (24-hydroxylase) which
converts 25OHD3 to the degradation product
24,25-(OH)2D3 (11). Accordingly, there has been
strong evidence presented that the loosely regulated extrarenal
1-hydroxylase correlates with the appearance of hypercalcemic
episodes and thus might be the cause of the hypercalcemia associated
with sarcoidosis, lymphoma, and perhaps even some types of solid tumors
(9, 12, 13). Underlying the findings to date was always the possibility
that the extrarenal 1-hydroxylase might be the product of a gene
different from that coding for the renal enzyme and therefore regulated
differently by the calcium homeostatic machinery.
The renal 1-hydroxylase enzyme is the result of a combination of the
activities of three proteins, a specific cytochrome P450 and two
general proteins, ferredoxin and ferredoxin reductase. Partially
purified preparations of the three proteins have been reconstituted to
give the 1-hydroxylase enzyme activity in vitro (14).
Very recently, the specific cytochrome P450 (CYP1), representing the
key protein of the renal 1-hydroxylase enzyme complex has been
cloned from rat (15) and subsequently from mouse and human (16, 17, 18, 19).
In vitro transfection studies of CYP1 together with
chromosomal analyses have indicated that this is indeed the gene
responsible for the renal production of
1,25-(OH)2D3 and is defective in the
hereditary form of rickets known as vitamin D dependency rickets type I
(15, 19, 20). Follow-up studies have identified response elements
within the 5'-flanking region of the rat CYP1 gene that allow it to
be up-regulated by PTH, but in these same studies researchers were
unable to identify elements responsible for down-regulation of the gene
by 1,25-(OH)2D3 (21). The recent
availability of the renal CYP1 sequence now allows for the first
direct molecular examination of CYP1 in extrarenal tissues. In the
present report, we provide evidence that the extrarenal CYP1 is a
product of the same gene as the renal form and is detectable in
vitro at both the messenger RNA (mRNA) and enzyme activity levels
in certain colon and lung cancer cell lines that we studied. We
speculate on the possible physiological role and importance of
extrarenal CYP1 in the pathological processes resulting in the
hypercalcemia of cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase-expressed sequence
tag (EST)-hydroxylase
(GenBank accession no. AB006034) was used to search the human EST
database for homologous sequences. Two human extrarenal complementary
DNAs (cDNAs) were found. EST 587798 (1.84 kb) from a
Stratagene (La Jolla, CA) colon library and EST 768387
(1.64 kb) from a Stratagene pancreas library. Both were
obtained and sequenced. EST 587798 was identical to the corresponding
portion of the human renal 1-hydroxylase (GenBank accession no.
AB005038), whereas EST 768387 was also identical, except that it
contained a portion of genomic 1-hydroxylase, similar to GenBank
accession no. AB005990.
Nonsmall cell lung carcinoma (NSCLC) cell lines
SK-MES-1, SW 900, Calu-1, SK-Luci-6, and WT-E were provided by
Dr. Barbara Campling. Cells were grown in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 5% FBS,
penicillin (50 U/ml), and streptomycin (50 µg/ml) at 37 C in an
atmosphere of 5% CO2 and 95% air.
Northern blot analysis
Poly(A)+ RNA was isolated from cultured cells using
the Oligotex Direct mRNA kit (QIAGEN, Valencia, CA) and
electrophoresed on a formaldehyde-agarose gel. The gel was photographed
under UV light and then blotted onto Hybond ECL nitrocellulose membrane
(Amersham, Arlington Heights, IL) and fixed to the
membrane by baking at 80 C for 2 h. Prehybridization and
hybridization were performed using QuikHyb (Stratagene). A
1590-bp restriction fragment from EST 587798 was labeled with
dATP[-32P] using the Prime-It II kit
(Stratagene). The blot was washed twice for 15 min in 2x
SSC (standard saline citrate)-0.1% SDS at room temperature, then for
15 min at 60 C in 0.1 x SSC-0.1% SDS and exposed at -70 C for
19 h to Kodak X-Omat AR film (Eastman Kodak Co.,
Rochester, NY).
The same blot was stripped and reprobed in a similar manner using a labeled 408-bp PCR product generated from a human CYP24 cDNA clone (22) donated by Dr. H.F. DeLuca, University of Wisconsin-Madison.
RT-PCR analysis of cDNA
Total RNA was isolated from NSCLC cultured cells using TRIzol
reagent (Life Technologies) followed by deoxyribonuclease
I (Life Technologies) treatment. RT was performed on 2
µg total RNA using an AMV reverse transcriptase kit according to the
manufacturer’s protocol (Promega Corp., Madison, WI). PCR
amplification was performed by mixing the following components (final
concentrations) on ice: 0.2 mM dNTPs, 1 mM
MgCl2, 1 x PCR buffer (QIAGEN), 0.5
pmol/µl upstream primer, 0.5 pmol/µl downstream primer, 5 µl RT
reaction, water to 49.5 µl, then 0.5 µl Taq DNA
polymerase (5 U/µl; QIAGEN) was added. Reaction
conditions were 1 cycle at 94 C for 2 min, followed by 35 cycles of 94
C for 30 sec, 58 C for 40 sec, and 72 C for 1.5 min, followed by a
final extension of 5 min at 72 C. The oligonucleotides for extrarenal
1-hydroxylase detection were based on the human renal
1-hydroxylase sequence (17). The upstream primer was
5'-ACCATGACCCAGACCCTCAAGTA-3', and the downstream oligonucleotide was
5'-CTCTGAGCAAATGCAAACATCTGG-3'. The upstream and downstream
oligonucleotides for CYP24 detection were
5'-AAACTAATGAAAC-CAGGGGAAGTG-3' and 5'-TCTGCACTAGGCTGCTGAGAATAC-3',
respectively. To control for contamination of PCR samples due to CYP24
or CYP1 cDNAs, independent control reactions were set up in which no
RT-generated cDNA was included. Ethidium bromide-stained gels showed an
absence of products when we did not add specific cDNA samples. This was
verified on the Southern analysis, which showed no hybridizing bands in
the control lanes.
Southern analysis of RT-PCR products
The RT-PCR products were electrophoresed and blotted onto
NitroPure nitrocellulose transfer membrane (Micron Separations, Inc.,
Westboro, MA). Hybridization was performed at 42 C using gene-specific
internal oligonucleotides, 5'-CTGCAGCTCGTGTAGCCTCGAC-3' for CYP1 and
5'-AAACGTGCTCATCATTGTTTTGAT-3' for CYP24. Oligonucleotides were end
labeled using ATP[
-32P] and T4 polynucleotide kinase.
The blots were washed and exposed at -70 C to Kodak X-Omat AR film
(Eastman Kodak Co.).
Induction of CYP24 activity by 25OHD3
SW 900 cells were cultured in RPMI 1640 supplemented with 5%
FCS. Approximately 8.5 x 106 cells were washed with
PBS twice, and the medium was replaced with RPMI 1640 supplemented with
1% BSA. The inducer 10 µM 25OHD3 and the
antioxidant 100 µM 1,2-dianilinoethane were added, and
the cells were incubated for 24 h. Total RNA was prepared using
TRIzol reagent (Life Technologies), treated with
deoxyribonuclease I, and reversed transcribed using an AMV reverse
transcriptase kit according to the supplier’s directions
(Promega Corp.). PCR amplification was performed in a
Perkin Elmer PCR machine as follows: 10 µl cDNA
synthesis reaction, 2.5 U Taq DNA polymerase
(Qiagen), 200 µM cDNA reaction dNTPs
(contributed by the first strand cDNA reaction), 2 mM
MgCl2, 1 x RT buffer (Promega Corp.),
and 1 µM of each upstream and downstream primer for CYP24
or CYP1. PCR conditions were as follows: 1 cycle at 94 C for 2 min;
35 cycles at 94 C for 30 s, 56 C for 40 s, and 72 C for 1
min; followed by a final extension for 5 min at 72 C. Aliquots (10
µl) of the PCR reactions were electrophoresed and photographed. PCR
reactions were controlled as previously described for RT-PCR analysis
of cDNA.
Cloning of the full-length 1-hydroxylase cDNA
The full-length PCR product from SW 900 was obtained using the
upstream primer 5'-GGCGGATCCAGGGGTTGAGATATGATGCTC-AGG-3' and the
downstream primer 5'-GACGAATTCTGGTCAGATAGGCATTAGGGGAAG-3' according to
the method outlined previously for RT-PCR. The product was purified
using the QIAquick PCR purification kit (QIAGEN) and
digested with EcoRI and BamHI. The gene was then
ligated into the pcDNA3.1(+) vector using T4 DNA ligase (Life Technologies), electroporated into competent Escherichia
coli, plated on Luria Bertoni-ampicillin plates, and incubated
overnight at 37 C. Colonies were grown up in Luria Bertoni-ampicillin
medium, and DNA was prepared using High Pure Plasmid Isolation Kit
(Boehringer Mannheim, Laval, Canada).
In vitro studies of vitamin D metabolism in cultured cells
Cells were grown to confluence in RPMI 1640 supplemented with
5% FCS on 100-mm plates, washed with PBS twice before the start of
metabolic studies to minimize the sequestrating effect of vitamin
D-binding globulin, present in the FCS, on 25OHD3 uptake by
cells. Medium was replaced with RPMI 1640 supplemented with 1% BSA to
act as a carrier of vitamin D and 100 µM
1,2-dianilinoethane, an antioxidant. Approximately 100,000 dpm
[26,27-3H]25OHD3 (Amersham; SA,
20 Ci/mmol) was premixed with medium containing the above additives,
then added to each plate of cells (4 ml/plate), and incubated for
24 h at 37 C in a 5% CO2 atmosphere. Negative
controls contained medium and radioactive substrate without cells. At
the end of the incubation period, methanol was added to stop the
enzymatic reaction and start the Bligh and Dyer lipid extraction
procedure as described previously (23). Sample preparation for HPLC
involved solubilization of the N2-dried extract in 115 µl
HPLC mobile phase, hexane-isopropanol-methanol in either 91:7:2 or
94:5:1 (vol/vol/vol).
In later experiments, confluent SW-900 cells were pretreated with 10 µM 25OHD3 to induce CYP24, and the cells were then washed with PBS to remove inducer, cultured with 70,000 dpm [26,27-3H]25OHD3, extracted, and prepared for HPLC as described above.
Analysis of vitamin D metabolites by HPLC
Straight phase LC was performed using a 2690 Alliance system
(Waters Corp., Milford, MA) equipped with a Zorbax-SIL
column and a diode array spectrophotometric detector recording in the
200–350 nm range. Solvent systems were mixtures of the solvents
hexane-isopropanol-methanol in either 91:7:2 or 94:5:1 (vol/vol/vol;
see figure legends for specific mixtures). Effluent was collected in
30-sec aliquots using a programmable fraction collector (Superrrac,
Pharmacia, Montreal, Canada), and radioactivity in dried fractions was
measured using a scintillation counter (Beckman Coulter, Inc., Palo Alto, CA).
In some experiments involving comigration of radioactive and
nonradioactive 1,25-(OH)2D3 standards, a
Berthold radioflow detector (model LB509, Wallac Inc.,
Turku, Finland) was used. In other experiments where it was necessary
to elute 1,24,25-(OH)3D3, a linear straight
phase gradient system was used from 91:7:2 to 88:10:2
hexane-isopropanol-methanol over 15 min starting at 0 min, followed by
a hold at 88:10:2 hexane-isopropanol-methanol for 5 min before a
reverse to starting conditions.
Reverse phase LC was performed using a 2690 Alliance system (Waters Corp.) equipped with a Zorbax-ODS column and a diode array spectrophotometric detector recording in the 200–350 nm range. A reverse phase gradient system was used from 50:50 to 100:0 acetonitrile-water over 25 min starting at 0 min, followed by a reverse to starting conditions. Effluent was collected in 15-sec aliquots using a programmable fraction collector (Superrrac, Pharmacia), and radioactivity in dried fractions was measured using an aqueous cocktail solution and a scintillation counter (Beckman Coulter, Inc.).
Transfection and enzyme activity of the full-length
1-hydroxylase cDNA in COS-1 cells
COS-1 monkey kidney cells were subcultured 16–20 h before
transfection and seeded in 100-mm plates at a density of 2 x
104 cells/cm2. Transfection was carried out
using the standard diethylaminoethyl-dextran procedure described in Guo
et al. (24). Cells were transfected with the mammalian
expression vector pcDNA3.1(+) containing full-length CYP1, as
constructed above, or the same pcDNA3.1(+) vector containing no insert.
Approximately 24 h after transfection, cells were subcultured into
six-well plates, and 24 h later 1-hydroxylase enzyme activity
was measured. For this, the transfected cells were washed twice with
PBS, and 1 ml unsupplemented DMEM containing 1% BSA and 50,000 cpm
[26,27-3H]25OHD3 was added to each plate.
After a 6-h incubation, the reaction was stopped by the addition of
methanol followed by extraction and chromatography as described above
for lung cell cultures.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase-hydroxylase (17). Subsequent RT-PCR analyses have confirmed
that this cell line contains transcripts for full-length CYP1 (data
not shown). The nucleotide sequence of this partial clone was 100%
identical over approximately two thirds of the published coding
sequence of the human renal enzyme. As this cell line was derived from
a putative secondary metastasis in a patient with a suspected primary
tumor of the lung, and as others had found 1-hydroxylase enzyme
activity in a lung cancer cell line (9), we focused upon screening
other lung cancer cell lines for 1-hydroxylase activity and CYP1
mRNA.
Correlation of 1-hydroxylase enzyme activity and CYP1 mRNA
expression
Screening of five NSCLC for 1-hydroxylase enzyme activity
revealed the results shown in Fig. 1
. Two
of the cell lines, SW 900 (Fig. 1D) and SK-Luci-6 (Fig. 1B), showed
specific production of a putative peak of
[26,27-3H]1,25-(OH)2D3 on HPLC,
which was absent in no cell control incubations (Fig. 1F). The peak of
SW 900-generated
[26,27-3H]1,25-(OH)2D3 comigrated
on straight phase HPLC using Zorbax-SIL and a radioactive monitor with
both synthetic commercially available
[26,27-3H]1,25-(OH)2D3
(Amersham) and
[1ß-3H]1,25-(OH)2D3,
synthesized in our laboratory (25) [retention times: SW 900-generated
[26,27-3H]1,25-(OH)2D3, 12.167
min; synthetic
[26,27-3H]1,25-(OH)2D3, 12.179
min; synthetic
[1ß-3H]1,25-(OH)2D3, 12.147
min]. Similarly, the SW 900-generated
[26,27-3H]1,25-(OH)2D3 and
synthetic 1,25-(OH)2D3 comigrated exactly on
reverse phase HPLC using Zorbax-ODS (retention time, 9.8 min). The peak
was absent in no cell and dead cell controls, indicating that it is not
an artifactual peak, such as 19-nor,10-keto-25OHD3 reported
previously in cell-free dilute protein solutions. In addition, the
other cell lines, particularly WT-E (Fig. 1E) and Calu-1 (Fig. 1A),
produced none of the
[26,27-3H]1,25-(OH)2D3, but,
instead, produced significant quantities of the alternative metabolite
24,25-(OH)2D3 as well as small amounts of other
side-chain oxidized products. When we used Northern analysis to examine
cells for CYP1 mRNA, we again found positive signals of the correct
size in only two of the five cells, with SW 900 showing the strongest
signal. Upon longer exposure of the blot, a signal could be observed in
SK-Luci-6 (Fig. 2). We have not probed
the blots with controls for mRNA quantification; however, all samples
were quantified by UV detection and verified visually on the gels
before blotting. Northern data were corroborated by performing RT-PCR
on the five cell lines; only SW 900 and SK-Luci-6 gave the expected
860-bp band that hybridized to a specific internal CYP1
oligonucleotide on Southern analysis (Fig. 3). Thus, there appears to be a
correlation between 1-hydroxylase enzyme activity and CYP1 mRNA
expression in the five NSCLC lines.
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-hydroxylase and 24-hydroxylase in the kidney, we chose
to examine the cell lines for CYP24 activity and mRNA expression. On
Northern analysis, the cell lines that showed high levels of CYP24
activity (WT-E and Calu-1) also gave strong positive signals for CYP24
mRNA (Fig. 4). In these cell lines, WT-E
shows a single transcript as denoted by the top arrow (Fig. 4). We note that Calu-1 shows two signals, one of the same size as in
WT-E and another slightly smaller. On RT-PCR, all cell lines were
positive for CYP24 (data not shown). The discrepancy between the
Northern analysis and RT-PCR is probably a reflection of the
sensitivity of the PCR analysis. Thus, it appears that there is a
correlation between the 24-hydroxylase enzyme activity measurements and
Northern analysis for CYP24 mRNA, although there is a hint of
24-hydroxylase activity and/or CYP24 mRNA signal in SK-Luci-6 and
SK-MES-1 cells.
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), whereas vehicle-treated cells
continued to produce 1,25-(OH)2D3 (Fig. 5B),
and there were no products in the no cell controls (Fig. 5C). This
result was confirmed by incubation of induced cells with 10
µM 25OHD3 as substrate, which resulted in the
formation of a detectable peak on HPLC with the retention time of
standard 24,25-(OH)2D3 and the UV spectrum of a
vitamin D (data not shown). The appearance of 24-hydroxylase enzyme
activity in SW 900 cells was accompanied by the coincidental detection
of CYP24 mRNA as a band of about 400 bp on RT-PCR (Fig. 6A), whereas in untreated or
vehicle-treated cells no CYP24 band was evident, and only CYP1 was
detectable as a band of around 860 bp. Interestingly, despite being
induced to begin to express CYP24, SW 900 cells continued to express
CYP1 mRNA 24 h after induction with vitamin D (Fig. 6B),
although these cells failed to show production of the
[26,27-3H]1,25-(OH)2D3
metabolite. However, these extracts did contain a radioactive peak that
comigrated with 1,24,25-(OH)3D3 on gradient
HPLC (data not shown), although it was not possible to tell whether
this metabolite had been produced from
[26,27-3H]1,25-(OH)2D3.
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-hydroxylase-hydroxylase and incubated with
[26,27-3H]25OHD3 produced a peak that
comigrated with 1,25-(OH)2D3 on straight phase
HPLC, whereas all transfection controls, including COS-1 cells
transfected with empty vector, nontransfected COS-1 cells, and no cell
controls, gave a flat baseline in the
1,25-(OH)2D3 region of the chromatogram (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase from a human
lung nonsmall cell carcinoma. It is clear that the expressed mRNA in
this (and probably other) lung tumor cell(s) is the same size as that
previously characterized in kidney and is the product of the same gene.
As expected, the full-length SW 900 lung cell-CYP1 transfected into
monkey kidney COS-1 cells gives rise to the same
[26,27-3H]1,25-(OH)2D3 metabolite
peak formed by SW 900 cells under basal culture conditions. The cloning
here of the human enzyme from lung and colon cancer cells together with
the data of Fu et al. for an extrarenal 1-hydroxylase of
keratinocyte origin (18) suggests that there is only one form of the
enzyme. It remains to be seen if the forms previously detected in
alveolar macrophage and placenta are also identical.
In our studies of the five lung cell lines, we found that there was a
good correlation of the expression of the mRNA for CYP1 (measured by
Northern analysis or RT-PCR) and the 1-hydroxylase enzyme activity
or, alternatively, correlation of the mRNA for CYP24 (measured by
Northern analysis) and the 24-hydroxylase enzyme activity. This
reciprocal relationship between the two enzyme activities has been
observed before, particularly for renal preparations of vitamin
D-deficient and vitamin D-replete animals (26), but the P450 sequences
now make it possible to confirm this at the mRNA level.
The findings of CYP1 expression in two lung cell lines, CYP24
expression in two others, and not much activity in the fifth cell line
indicate inherent differences between the cell lines. It should be
noted that all five small cell carcinomas were cultured under identical
conditions. Thus, basal ionic, vitamin D, or other hormone conditions
should be identical, these being known factors in stimulating
1-hydroxylase or 24-hydroxylase expression. However, the abilities
of cell lines to produce and secrete autocrine or paracrine factors,
such as PTH-related peptide (PTHrP) or cytokines
(e.g. interferon-), probably differs between the
different cell lines, and these may play a role in CYP expression (27).
Other possible mechanisms modulating cytochrome P450 expression may
include growth factor or peptide hormone receptor expression.
Our experiments have revealed that under basal conditions, the SW 900
cell line expresses CYP1 mRNA and shows 1-hydroxylase enzyme
activity, whereas in the same cells treated with exogenous vitamin D
metabolites we observed expression of CYP24 mRNA and the synthesis of
24-hydroxylated metabolites. This switchover is similar to the process
observed in the kidney (26). Thus, vitamin D seems to be a stronger and
more overwhelming effector than the presumed unknown modulator that
pushes the SW 900 cell line into constitutive CYP1 expression.
Interestingly, RT-PCR using probes for CYP1 continues to show a
signal of the correct size for CYP1 expression 24 h after the
treatment with vitamin D and long after CYP24 has been induced, but the
production of 1,25-(OH)2D3 on HPLC is no
longer detectable. There may be several possible explanations for the
apparently conflicting data including the selective further metabolism
of 1,25-(OH)2D3. In fact, experiments
performed here demonstrate the formation of
1,24,25-(OH)3D3 after CYP24 appears in SW900
cells, and it is attractive to speculate that it is formed from
1,25-(OH)2D3. Certainly,
1,25-(OH)3 is known to be an excellent substrate for
CYP24 (28) and, if formed, would probably be quickly and preferentially
converted to C-24 oxidation products such as
1,24,25-(OH)3D3. The interesting phenomenon
is the continued expression of CYP1 mRNA 24 h after treatment
despite the presence of the 1,25-(OH)2D3
signal, which would be expected to turn off CYP1 expression based
upon earlier in vivo findings (29). However, the results
here and the recent report of no discernible effect of
1,25-(OH)2D3 on basal or PTH-induced
expression of a rat CYP1 promoter-driven reporter gene in
vitro (21) suggest that the previously observed suppressive action
of 1,25-(OH)2D3 in vivo must be
at a posttranscriptional level or is indirect and requires the presence
of some other agent absent from our in vitro model.
Another important implication of our work is the role of extrarenal
1-hydroxylase in the pathogenesis of the hypercalcemia of cancer.
All cell lines studied here were obtained randomly from tumor banks and
therefore presumably reflect the incidence of CYP1 expression in the
general pool of such tumors. This incidence is surprisingly high
compared with that observed by Mawer et al. (9) and implies
that the extrarenal production of
1,25-(OH)2D3 may be more common than is
currently believed, and part of the reason that this has not been
reported more frequently reflects the technical difficulty that existed
for proving the presence and activity of CYP1.
PTHrP production by the lung cell lines studied here is also unknown,
but is currently under investigation. The complex interrelationship of
1,25-(OH)2D3 and PTH/PTHrP, in which
1,25-(OH)2D3 down regulates PTH/PTHrP
synthesis and PTH/PTHrP up-regulates the CYP1 gene, suggests that a
paracrine loop may exist in extrarenal tissues for the regulation of
cell growth/differentiation, a hypothesis put forward previously (12, 30). The demonstration of cancer cell lines in which extrarenal
1,25-(OH)2D3 production is constitutive but
subject to weak regulation makes it possible to pursue the study of the
molecular events underlying the role of the extrarenal 1-hydroxylase
in this possible feedback loop.
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Footnotes |
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Received August 11, 1998.
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  R. J. Menezes, R. T. Cheney, A. Husain, M. Tretiakova, G. Loewen, C. S. Johnson, V. Jayaprakash, K. B. Moysich, R. Salgia, and M. E. Reid Vitamin D Receptor Expression in Normal, Premalignant, and Malignant Human Lung Tissue Cancer Epidemiol. Biomarkers Prev., May 1, 2008; 17(5): 1104 - 1110. [Abstract] [Full Text] [PDF]
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 S. Sundaram, M. J. Beckman, A. Bajwa, J. Wei, K. M. Smith, G. H. Posner, and D. A. Gewirtz QW-1624F2-2, a synthetic analogue of 1,25-dihydroxyvitamin D3, enhances the response to other deltanoids and suppresses the invasiveness of human metastatic breast tumor cells. Mol. Cancer Ther., November 1, 2006; 5(11): 2806 - 2814. [Abstract] [Full Text] [PDF]
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 M. van Driel, M. Koedam, C. J. Buurman, M. Hewison, H. Chiba, A. G. Uitterlinden, H. A. P. Pols, and J. P. T. M. van Leeuwen Evidence for auto/paracrine actions of vitamin D in bone: 1{alpha}-hydroxylase expression and activity in human bone cells FASEB J, November 1, 2006; 20(13): 2417 - 2419. [Abstract] [Full Text] [PDF]
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W. Zhou, R. Suk, G. Liu, S. Park, D. S. Neuberg, J. C. Wain, T. J. Lynch, E. Giovannucci, and D. C. Christiani Vitamin D Is Associated with Improved Survival in Early-Stage Non-Small Cell Lung Cancer Patients Cancer Epidemiol. Biomarkers Prev., October 1, 2005; 14(10): 2303 - 2309. [Abstract] [Full Text] [PDF]
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 B. Diesel, J. Radermacher, M. Bureik, R. Bernhardt, M. Seifert, J. Reichrath, U. Fischer, and E. Meese Vitamin D3 Metabolism in Human Glioblastoma Multiforme: Functionality of CYP27B1 Splice Variants, Metabolism of Calcidiol, and Effect of Calcitriol Clin. Cancer Res., August 1, 2005; 11(15): 5370 - 5380. [Abstract] [Full Text] [PDF]
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 K. Mimori, Y. Tanaka, K. Yoshinaga, T. Masuda, K. Yamashita, M. Okamoto, H. Inoue, and M. Mori Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer Ann. Onc., February 1, 2004; 15(2): 236 - 241. [Abstract] [Full Text] [PDF]
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 S. Komuro, M. Sato, and H. Kanamaru DISPOSITION AND METABOLISM OF F6-1{alpha},25(OH)2 VITAMIN D3 AND 1{alpha},25(OH)2 VITAMIN D3 IN THE PARATHYROID GLANDS OF RATS DOSED WITH TRITIUM-LABELED COMPOUNDS Drug Metab. Dispos., August 1, 2003; 31(8): 973 - 978. [Abstract] [Full Text] [PDF]
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 U. Segersten, P. Correa, M. Hewison, P. Hellman, H. Dralle, T. Carling, G. Akerstrom, and G. Westin 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase Expression in Normal and Pathological Parathyroid Glands J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2967 - 2972. [Abstract] [Full Text] [PDF]
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R. M. Maas, K. Reus, B. Diesel, W.-I. Steudel, W. Feiden, U. Fischer, and E. Meese Amplification and Expression of Splice Variants of the Gene Encoding the P450 Cytochrome 25-Hydroxyvitamin D3 1,{{alpha}}-Hydroxylase (CYP 27B1) in Human Malignant Glioma Clin. Cancer Res., April 1, 2001; 7(4): 868 - 875. [Abstract] [Full Text]
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