From The Texas A&M University System Health Science Center, Department of Pathology and Laboratory Medicine, College Station, Texas 77843-1114
Received for publication, September 30, 2002, and in revised form, December 20, 2002
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ABSTRACT |
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Proline oxidase is a p53-induced gene that can
mediate apoptosis in lung carcinoma cells. Here, we provide
evidence implicating a role for proline oxidase in renal carcinoma. We
observed absent or reduced expression of proline oxidase in 8 of 12 primary renal cell carcinomas, with respect to their normal tissue
counterparts. Two renal cell carcinomas, which displayed little or no
expression of proline oxidase, expressed p53s that were less capable of
inducing proline oxidase than p53 isolated from normal renal tissue.
One of those tumor-derived p53s contained a double transition mutation at amino acid residues 125 (Ala to Thr) and 193 (Arg to His), and the
other exhibited a single transition mutation at amino acid 149 (Ser to
Phe). Forced up-regulation of proline oxidase induced the formation of
reactive oxygen species and mediated apoptosis in the 786-0 renal cell
carcinoma cell line. A proline oxidase antisense vector repressed
p53-induced up-regulation of proline oxidase, release of cytochrome
c from mitochondria, and apoptosis in 786-0 renal
carcinoma cells. Taken together, these findings support a role for
proline oxidase as a downstream effector in p53-mediated apoptosis. We
hypothesize that its altered expression can contribute to the
development of renal carcinomas. The presence of proline oxidase
in mitochondria, a primary organelle that regulates apoptosis, places
this molecule in a subcellular localization that can directly influence
the apoptotic pathway and thus tumorigenesis.
The product of the p53 gene plays pivotal roles in several
biological processes that are important in reducing the
tumorigenic potential of cells. The protein mediates cell growth arrest
by controlling cell cycle checkpoints (1), induces apoptosis (2), and
can influence the differentiation state of cells (3). Many studies have
implicated p53 in genomic stability, surveillance of DNA damage, and
DNA repair, which are related to its control over cell growth
(4-9).
A series of p53-induced genes has been identified by a serial
analysis of gene expression in a colon carcinoma cell line; some of
those genes are predicted to encode proteins that could generate or
respond to oxidative stress (10). We performed a comparative gene
expression analysis between p53-sensitive and -resistant cells, which
also revealed the close association of redox enzymes with p53-mediated
apoptosis (11). One of those redox enzymes, proline oxidase, and its
product, pyrroline-5-carboxylate (P5C),1 exhibited the ability
to inhibit cell growth and to induce marked apoptosis in a lung
carcinoma cell line, implicating a role for the proline/P5C cycle in
p53-mediated growth inhibition and apoptosis (11). Our work was
supported by a later study showing that proline oxidase can generate
reactive oxygen species (12), which can initiate apoptosis by directly
acting on the mitochondrial permeability core complex and effecting the
mitochondrial permeability transition (13-15). Here, we report that
the expression of proline oxidase was absent or reduced in a
significant number of renal cell carcinomas, which was associated with
mutant p53 in two cases. A correlation of reduced proline oxidase
expression with mutated p53 in two cases of renal carcinoma and the
ability of proline oxidase to induce apoptosis in renal carcinoma cells
functionally implicate the enzyme as a downstream player in
p53-mediated apoptosis.
Antibodies and Plasmids--
The p53 monoclonal antibody,
Bp53-12, was purchased from Santa Cruz Biotechnology, the M30 antibody
specific for the caspase-cleaved form of cytokeratin 18 (16) from Roche
Molecular Biochemicals, a monoclonal antibody for cytochrome
c (C-8) from Santa Cruz Biotechnology, and an actin
monoclonal antibody (N350) from Amersham Biosciences. A proline oxidase
antibody (T338) was generated in rabbits against a synthetic peptide
composed of amino acids 338 through 353 of the proline oxidase protein
(TGQLEPLLSRFTEEEE). Kidney p53 cDNAs isolated by
reverse-transcription PCR from primary kidney tissues were cloned into
the pcDNA3.1 ECHO expression system (Invitrogen). The complete
proline oxidase cDNA was cloned into the pAdtrack vector (17),
which coexpresses GFP as a normalization control for transfection
efficiency. The pAdtrack-proline oxidase vector was used to generate
recombinant adenovirus using a simplified procedure as previously
described (17). A proline oxidase antisense vector was constructed by
subcloning the proline oxidase cDNA encoding amino acids 186-516
in the pEGFPC2 expression vector (Clontech) in the
antisense orientation. The proline oxidase and a wild type p53
recombinant adenovirus were amplified in 293 cells and virus-containing
medium used to infect kidney cells.
Cell Culture, Transfection, Western Blotting, and Proline Oxidase
Enzyme Assays--
The 786-0 renal carcinoma and the H1299 nonsmall
cell lung carcinoma cell lines were obtained from the American Tissue
Type Collection and propagated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. To conduct transfections of renal and
lung carcinoma cells, a T75 flask of confluent cells was incubated for
2 h at 37 °C in 3 ml of Optimem medium (Invitrogen) containing 4 µg of plasmid DNA and 3 µl of LipofectAMINE-2000 (Invitrogen). The transfection medium was then replaced with Dulbecco's modified Eagle's medium and the cells incubated for 24-36 h to allow
expression of the transfected gene. To normalize for transfection
efficiency, pAdtrack, which expresses green fluorescence protein, was
included in the transfection assay where appropriate. Western blotting was performed by using the SuperSignal immunodetection system (Pierce
Chemical), as previously described (18). Proline oxidase activity was
detected by the reduction of the colorimetric indicator 2,6-dicholoroindophenol (an electron acceptor) as a result of oxidation
of proline in extracts of isolated mitochondria, as previously
described (19). Mitochondria were isolated by homogenization of cells
in a 0.25 M sucrose buffer (20). After microcentrifugation of whole-cell lysates, the mitochondria-enriched pellet was washed twice in sucrose buffer.
Apoptosis Assays--
Flow cytometry to quantitate apoptosis was
performed on ethanol-fixed and permeabilized cells that were stained
with propidium iodide according to a previously published protocol
(11). Apoptotic cells were quantitated in the subG1 DNA content portion
of the flow cytometric scans. Reactive oxygen species were detected by the oxidation of dihydroethidium in the cytoplasm of viable cells. The
oxidized compound then migrated to the nucleus as ethidium bromide and
emitted a red-orange fluorescence under ultraviolet light when bound to
DNA (21). Early apoptosis was detected by immunohistochemistry using a
monoclonal antibody (M30) specific for the caspase-cleaved form of
cytokeratin 18 (16, 22), according to the manufacturer's
specifications (Roche Molecular Biochemicals). Apoptotic cells were
also identified by morphological criteria that included cell blebbing,
fragmented and shrunken nuclei, and apoptotic bodies.
Tumor cDNA Blotting--
Paired normal/tumor cDNA blot
arrays were purchased from Clontech. The cDNA
blot was hybridized with a proline oxidase cDNA probe according to
the manufacturer's specifications and then rehybridized with an
ubiquitin probe for the purpose of normalizing the expression data.
Reverse-transcription Polymerase Chain Reaction
(RT-PCR)--
Paired normal and carcinoma cDNAs generated from
kidney tissues were purchased from Clontech. RT-PCR
on nonsmall cell lung and renal carcinoma cell lines was performed by
isolating total RNA using the TRI reagent extraction solution
(Molecular Research Center, Inc.) and generating cDNA from the RNA
by using the RT-for-PCR system (Clontech).
RT-PCR to analyze p53, proline oxidase, Bax, p21waf1/cip1, and glyceraldehyde-3-phosphate dehydrogenase
expression was performed as previously described (11). Ribosomal
protein S9 primers for normalization of PCR were purchased from Clontech.
Proline Oxidase Expression Is Absent or Reduced in Many Renal Cell
Carcinomas--
A commercial array containing cDNA amplified from
paired normal and tumor mRNA from 12 patients presenting with renal
carcinoma was hybridized with a 32P-labeled proline oxidase
cDNA probe (Fig. 1, upper
panel). The cDNA was normalized by rehybridizing the blot with
a ubiquitin housekeeping cDNA probe (Fig. 1, lower
panel). The cDNA spotted on the membrane has been shown to
correlate closely with the expression of a number of different known
mRNAs in the tissue (Clontech). As shown in
Fig. 1, five kidney tumors (POX panel, T row,
lanes 3, 4, 5, 7,
10) appeared to exhibit little or no expression of proline
oxidase mRNA as compared with the matched normal tissue (N
row), even though similar amounts of normal and tumor cDNA were present on the blot as shown by rehybridization of the blot with a
ubiquitin housekeeping cDNA probe (Fig. 1, Ubiq.). When taking the ubiquitin normalization blot into consideration, it appeared
that several other renal cell carcinomas exhibited reduced expression
of proline oxidase mRNA (POX panel, T row,
lanes 1, 8, 9). Reduced or absent
proline oxidase expression thus occurred in two-thirds of the
renal cell carcinomas that we examined. This tumor array data suggested
that altered expression of proline oxidase is frequently associated
with renal cell carcinomas. However, to validate the differential
expression of proline oxidase between normal and tumor tissues that we
observed in the tumor array cDNA blot, the cDNA derived from
kidney 4 and kidney 7 normal and tumor tissues was subjected to
PCR. As shown in Fig. 2, the expected 270-bp-size proline oxidase DNA product was generated from normal kidney tissue (N lanes) cDNA, whereas little or no
product was derived from tumor tissue cDNA (T lanes),
confirming the array data showing that kidney tumors 4 and 7 lacked
apparent expression of proline oxidase. In both renal cell carcinomas 4 and 7, the reduced expression of proline oxidase was very specific,
because transcripts for p53, p21waf1/cip1, and Bax were
expressed at similar levels in both normal and tumor tissues, relative
to the ribosomal S9 protein that was used as a normalization control.
From an analysis of the classification and staging of these renal
carcinomas, it appeared that reduced expression of proline oxidase was
more prevalent in clear cell-type renal cell carcinomas at stages II to
IV (Table I; tumors 1, 7, 8, 9, 10).
However, two clear cell-type carcinomas did display normal
levels of proline oxidase (Table I; tumors 6,
11). No differences in expression of proline oxidase were
detected by the tumor array blotting method in three prostate
carcinomas relative to their normal tissue counterparts (data not
shown).
Up-regulation of Proline Oxidase Induces Apoptosis in Renal
Carcinoma Cells--
To determine the biological function of proline
oxidase in renal cell carcinoma, the 786-0 renal carcinoma cell line
was transfected with the proline oxidase expression vector
(pAdtrack-Pox-1) to up-regulate proline oxidase. Proline oxidase
expression was determined by RT-PCR, Western blotting, and a
colorimetric assay to detect oxidation of proline in cell lysates. As
shown in Fig. 3A, lane 3, little or no expression of proline oxidase mRNA was
detected in 786-0 cells transfected with the pAdtrack-GFP expression
vector, whereas considerable levels of proline oxidase transcripts were detected in cells transfected with the pAdtrack-Pox-1 vector (Fig. 3A, lane 4). Considerable induction of proline
oxidase protein was observed in 786-0 cells 24 h after
transfection with the pAdtrack-POX vector (Fig. 3C,
lane 3) or infection with two different isolates of
recombinant proline oxidase adenovirus (Fig. 3C, lanes
1 and 2). No proline oxidase protein was detected in
786-0 cells infected with a recombinant GFP adenovirus (Fig.
3C, lane 4). To measure proline oxidase activity,
we relied on a method utilizing the reduction of the colorimetric
indicator 2,6-dicholoroindophenol (DIC), an electron acceptor, as a
result of oxidation of proline in extracts of isolated mitochondria
(19). Reduction of DIC results in a decrease in absorbance at 600 nm
over time. As shown in Fig. 3B, the reduction of DIC
occurred to a significantly higher degree in mitochondrial extracts
from pAdtrack-Pox-1-transfected cells than in mitochondrial extracts
obtained from pAdtrack-GFP cells. Lysates of pAdtrack-GFP-only
transfected cells were used as a blank for the absorbance.
Forced overexpression of p53 in 786-0 cells by infection with the
pcDNA3.1-wild type p53 expression vector or with the recombinant p53 adenovirus induced apoptosis (Fig. 6A) and up-regulated
the proline oxidase protein (Fig.
4A, mitochondria,
lane 3). Expression of p53 was easily observed by Western
blotting in 786-0 cells infected with p53 adenovirus, in contrast to
the apparent absence of p53 protein in GFP-infected cells (Fig.
4A, whole-cell extract, lower left
panel). Essentially all of the induced proline oxidase protein was
observed in the mitochondria (Fig. 4A,
mitochondria, lane 3) with little or no enzyme
located in the cytoplasm (cytoplasm panel, lane
3). Forced up-regulation of proline oxidase by transfection with
pAdtrack-POX resulted in significant increases in apoptosis in 786-0
cells, as indicated by the increases in the subG1 population in flow
cytometric scans relative to those in GFP-transfected cells (Fig.
4B).
Proline oxidase has been shown to induce the formation of reactive
oxygen species (12). Dihydroethidium localizes in the cytoplasm and,
when oxidized by reactive oxygen, binds DNA and emits an orange-red
fluorescence under UV light. As shown in Fig. 4C, proline
oxidase-transfected 786-0 cells treated with dihydroethidium showed
considerably more orange-red nuclear fluorescence than GFP-only
transfected cells, indicating that proline oxidase induced the
generation of reactive oxygen species. Fig. 4C shows the
condensed and fragmented nuclear morphology characteristic of the
latter stages of apoptosis in 786-0 cells.
Caspase-mediated cleavage of cytokeratin 18 is an event associated with
the early stages of apoptosis (22). An antibody (M30) specific for an
epitope that is uncovered only in caspase-cleaved forms of cytokeratin
18 can be used as a marker of early apoptosis (16).
Immunohistochemistry showed that 786-0 cells expressing both GFP and
proline oxidase were specifically reactive with the primary M30
monoclonal antibody, as detected by the orange-red fluorescence emitted
by the phycoerythrin-conjugated secondary antimouse immunoglobulin
(Fig. 5A, right
panels). Little or no orange-red fluorescence was detected in
GFP-only transfected cells (Fig. 5B, right
panel).
A direct role for proline oxidase in p53-mediated apoptosis was
suggested from proline oxidase antisense expression studies. Forced
up-regulation of wild type p53 in 786-0 cells by transfection with the
pcDNA3.1-wtp53 expression vector induced considerable apoptosis
36 h after transfection (48%) (Fig.
6A). Cotransfection of these
cells with wild type p53 and a proline oxidase antisense vector
resulted in significantly less (18%) apoptosis (Fig. 6A, p53+pox(antisense)) and suppressed expression of proline
oxidase protein by up to 80% (Fig. 6B, compare lane
2 with lane 1). Moreover, the proline oxidase antisense
vector prevented p53-mediated release of cytochrome c from
mitochondria (Fig. 6C, compare lane 3 with lane 2).
Reduced or Absent Expression of Proline Oxidase in Two Renal Cell
Carcinomas Is Associated with Mutated p53--
Our published work (11)
and data presented here indicate that proline oxidase is a proapoptotic
p53-induced gene whose expression is frequently reduced or absent in
renal cell carcinomas. We asked whether the altered expression of
proline oxidase in kidney tumors might be associated with mutant p53
proteins. To determine the functionality of kidney tumor-derived p53,
we cloned the p53 cDNAs from both normal and tumor tissue of
kidneys 4 and 7. These p53 cDNAs were cloned into the ECHO
expression system to determine whether they were capable of inducing
proline oxidase in p53-null H1299 lung carcinoma cells. As shown in
Fig. 7A, Western blotting demonstrated that p53 cDNAs isolated from normal and tumor kidney tissues were efficiently expressed in H1299 cells (lanes
2-4). Reverse-transcription PCR revealed that p53 isolated from
normal kidney 4 dramatically induced the expression of proline oxidase in H1299 cells (Fig. 7B, lower level, compare
lane 6 with lane 5). Up-regulation of the p53s
from tumors 4 and 7 also resulted in the induction of proline oxidase
transcription, albeit to considerably lower levels than that induced by
the normal kidney p53 (Fig. 7B, compare lanes 7 and 8 with lane 6). Western blotting showed that
the levels of expression of proline oxidase protein correlated closely
with the expression of mRNA detected by RT-PCR (Fig. 7A, lanes 2-4). Because both p53 protein and mRNA levels
were approximately equivalent between the normal and tumor
p53-transfected cells and proline oxidase mRNA and protein were
differentially up-regulated by normal and tumor-derived p53s, we
surmised that the tumor-derived p53s were less efficient at
transactivating the proline oxidase gene. Sequencing of the p53
cDNAs was thus performed in an attempt to derive a structural
explanation for the differential induction of proline oxidase between
these normal and tumor-derived p53s. The p53 isolated from renal cell
carcinoma 4 contained a double transition mutation at amino acid
residues 125 (Ala to Thr) and 193 (Arg to His), and that cloned from
tumor 7 exhibited a single transition mutation at amino acid 149 (Ser
to Phe).
We conclude that proline oxidase is a downstream mediator in
p53-mediated apoptosis in renal carcinomas, based on a number of
findings presented in this manuscript. First, up-regulation of p53
induced the expression of the proline oxidase gene. Second, a proline
oxidase antisense expression vector suppressed p53-mediated apoptosis
in a renal carcinoma cell line. Third, two renal tumor-derived p53
mutants were less efficient in inducing the proline oxidase gene than
wild type p53 isolated from normal renal tissue. Fourth, forced
expression of proline oxidase induced substantial apoptosis in a renal
carcinoma cell line. Finally, proline oxidase can generate reactive
oxygen species, which mediate apoptosis by influencing mitochondrial
membrane potential by acting on the mitochondrial membrane permeability
core complex (13-15).
Although the exact mechanism by which proline oxidase mediates
apoptosis is not known, the enzyme might facilitate apoptosis through
products generated from the proline redox cycle. Proline oxidase
catalyzes the conversion of proline to P5C and transfers electrons into
the mitochondrial electron transport with an intervening flavoprotein
(20). The pair of electrons donated by proline can generate two ATP
molecules. P5C is then shuttled outside the mitochondria where it is
converted to proline by P5C reductase, altering the NAD/NADP oxidation
balance. The NAD oxidation state has been shown to be important in the
regulation of cell growth (23) and apoptosis (24). Moreover, proline
oxidase can generate reactive oxygen species (12), which can effect the
mitochondrial membrane permeability transition (13-15).
In summary, our data implicate a role for altered expression of proline
oxidase in renal cell tumorigenesis. The data also demonstrate a direct
role for proline oxidase in p53-mediated apoptosis of renal carcinoma
cells. We have also observed alterations in proline oxidase expression
in primary lung carcinomas, lung carcinoma cell
lines, and a primary stomach
cancer,2 suggesting a role for this redox enzyme in
other types of cancers as well.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Proline oxidase expression is absent or
reduced in renal carcinomas as determined by cDNA tumor array
analysis. Relative expression of proline oxidase was determined in
normal (N) and tumor (T) tissue by hybridizing
the cDNA tumor array blot with a proline oxidase cDNA probe
(upper panel). The tumor array blot was rehybridized with an
ubiquitin cDNA probe for normalization of cDNA (lower
panel). Twelve different renal cell carcinomas (1-12)
were analyzed.
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Fig. 2.
RT-PCR confirmed altered expression of
proline oxidase in renal carcinomas. RT-PCR was performed on two
renal cell carcinomas (4 and 7) that displayed
reduced or absent proline oxidase expression in the tumor array
analysis shown in Fig. 1. Ribosomal protein S9 was employed as a
normalization control. The expression of p53 and two of its target
genes (Bax and p21) was also investigated in the
same experiment.
Renal cell carcinoma and proline oxidase expression
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Fig. 3.
Forced expression of proline oxidase in renal
carcinoma cells. Proline oxidase cDNA was cloned into the
pAdtrack adenovirus expression vector and transfected into 786-0 renal
carcinoma cells. Expression of proline oxidase was determined by
RT-PCR (A), by an enzyme assay to detect proline-specific
oxidation (B), and by Western blotting using a rabbit
antibody that was generated against a proline oxidase peptide
(C). A, total RNA was isolated from 786-0 cells
transfected with pAdtrack-GFP (lane 3) or pAdtrack-proline
oxidase (lane 4) expression vector and was subjected to
RT-PCR using a combination of glyceraldehyde-3-phosphate dehydrogenase
and proline oxidase primers. Lanes 1 and 2,
control assays of pAdtrack-POX-transfected 786-0 cells using only
glyceraldehyde-3-phosphate dehydrogenase or proline oxidase primers,
respectively. Lanes 5 and 6, RT-PCR assays of
kidney 4 normal and tumor tissues, respectively, as additional controls
for proline oxidase expression. B, proline oxidase-specific
reduction of DIC in lysates of 786-0 cells 24 h after
transfection with pAdtrack-POX. The reduction of DIC was reflected in
the corresponding decrease in absorbance of the reduced DIC at 600 nm.
Values obtained from proline oxidase-transfected cell lysates were
subtracted from those determined from GFP-transfected lysates to detect
proline oxidase-specific activity. C, forced expression of
proline oxidase protein in 786-0 cells. Whole-cell lysates were
prepared from 786-0 cells infected with two different isolates of
recombinant proline oxidase (POX) adenovirus (lanes
1 and 2) or GFP adenovirus (lane 4) or
transfected with the pAdtrack-POX vector (lane 3). Proteins
were extracted 24 h after infection or transfection and were
immunoblotted for proline oxidase expression by using the T338 proline
oxidase peptide antibody. GFP- and proline oxidase
(POX)-transfected lysates were also immunoblotted for actin
as a protein loading control.
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Fig. 4.
Up-regulation of proline oxidase induces
apoptosis and reactive oxygen species in 786-0 renal carcinoma
cells. A, p53-mediated induction of proline oxidase in
786-0 renal carcinoma cells. 786-0 cells were mock-infected
(lanes 1) or infected with GFP (lanes 2) or p53
recombinant adenovirus (lanes 3). Mitochondria were isolated
24 h after transfection, and cytoplasmic proteins in the
postmitochondrial supernatant were acetone-precipitated. Proteins in
mitochondrial and cytoplasmic pellets were solubilized in SDS buffer
and analyzed for proline oxidase expression in Western blots by using
the T338 proline oxidase antibody. Whole-cell extracts were also
prepared by lysing 786-0 cells infected with recombinant GFP and p53
adenoviruses in SDS gel-loading buffer. The extracts were immunoblotted
for proline oxidase (POX), p53, or actin (as a protein
loading control). B, 786-0 cells were transfected with
pAdtrack-GFP or pAdtrack-proline oxidase. 24 h later, apoptotic
cells were quantitated by flow cytometry of ethanol-fixed and propidium
iodide-stained cells. C, generation of reactive oxygen
species in GFP-only and proline oxidase-transfected 786-0 cells. Cells
were infected with GFP or proline oxidase recombinant adenovirus for
12 h and then incubated with dihydroethidium, which when oxidized
by reactive oxygen binds to DNA as ethidium, emitting an orange-red
fluorescence under ultraviolet light. Generation of reactive oxygen was
then quantitated by flow cytometry of GFP and ethidium fluorescence.
Proline oxidase-induced cells showed considerably more
ethidium-staining than GFP-only cells, indicating the increased
generation of reactive oxygen species. D, a
dihydroethidium-stained proline oxidase-transfected cell (24 h after
transfection) showing nuclear condensation and fragmentation that is
characteristic of the later stage of apoptosis.
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Fig. 5.
Immunohistochemistry of proline
oxidase-transfected 786-0 cells using M30 antibody specific for the
caspase-cleaved form of cytokeratin 18. A, detection of
caspase-cleaved forms of cytokeratin-18 in proline oxidase-transfected
786-0 cells. The left panels show only the green
fluorescence emitted by GFP, and the right panels show only
the orange-red fluorescence emitted by the phycoerythrin secondary
antibody, using the appropriate filters. Numerous cells transfected
with proline oxidase exhibited orange-red staining, indicating
activation of caspase activity and cleavage of cytokeratin 18. B, little or no M30 staining was observed in GFP-transfected
786-0 cells (right panel).
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Fig. 6.
A proline oxidase antisense vector suppressed
p53-mediated apoptosis of 786-0 renal carcinoma cells.
A, 786-0 cells were transfected with GFP, p53, or p53 and a
proline oxidase antisense expression vector (p53+POX(antisense)).
After 36 h, cells were harvested, fixed, and prepared for
flow cytometry. Apoptotic cells were quantitated in the subG1 region of
the flow cytometric scan. B, cotransfection of p53 and
POX(antisense) expression vectors resulted in down-regulation of the
amount of proline oxidase induced in 786-0 cells (lane 2)
relative to cells cotransfected with p53 and GFP (lane 1).
786-0 cells were transfected and subjected to Western blotting by
using the T338 antibody as described under "Materials and Methods."
C, release of cytochrome c from mitochondria by
p53 (lane 2) is suppressed by the proline oxidase antisense
(lane 3). Lane 1, a GFP-transfected control.
Postmitochondrial supernatant proteins were acetone-precipitated and
subjected to Western blotting using the C-8 cytochrome c
monoclonal antibody. The cytochrome c blot was stripped and
reprobed with an actin monoclonal antibody to normalize for protein
loading.
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Fig. 7.
Two renal cell carcinomas expressed mutant
p53s that were less efficient in transactivating the proline oxidase
(POX) gene than a wild type p53 derived from normal
kidney tissue. A, p53 cDNAs were cloned from normal
renal tissue and renal carcinomas 4 and 7 into the ECHO expression
system. Vectors were transfected into the p53-null H1299 nonsmall cell
lung carcinoma cell line for 24 h. Proline oxidase
(POX) and p53 expression were investigated in transfected
cells by Western blot using T338 peptide antibody and Bp53-12
monoclonal antibody, respectively. Lane 1, GFP-transfected;
lane 2, normal kidney p53; lane 3, renal
carcinoma 4 p53; lane 4, renal carcinoma 7 p53.
B, RT-PCR analysis of the expression of p53 and proline
oxidase (POX, lanes 5-8) and ribosomal protein
S9 (lanes 1-4) as a normalization control. Lanes
1 and 5, GFP-transfected; lanes 2 and
6, normal kidney p53; lanes 3 and 7,
tumor kidney 4 p53; lanes 4 and 8, tumor kidney 7 p53. Transcript expression for p53 and proline oxidase (B)
correlated well with that observed for the respective proteins
(A).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: E-mail:
s-maxwell@tamu.edu.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M210012200
2 S. Maxwell, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: P5C, pyrroline-5-carboxylate; GFP, green fluorescence protein; RT, reverse transcription; DIC, 2,6-dicholoroindophenol; POX, proline oxidase.
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