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Experimental Therapeutics |
Department of Molecular Biology, at the University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, New Jersey 08084 [R. D. L., S. J. C.]; QualTek Molecular Laboratories, Inc., Santa Barbara, California 93111 [F. J. L.]; and Division of Medical Oncology, University of Southern California/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033 [S. G., Y. P. X., A. S., J. S., H-J. L.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using a monoclonal antibody (MAb), DUT415, that cross-reacts with both nuclear and mitochondrial isoforms of human dUTPase. Nuclear and cytoplasmic staining was observed in both normal and neoplastic tissues. In normal tissues, nuclear dUTPase staining was observed exclusively in replicating cell types. This observation is in agreement with cell culture studies where expression of the nuclear isoform (DUT-N) is proliferation dependent. In contrast, cytoplasmic expression of dUTPase does not correlate with proliferation status and was observed in tissues rich in mitochondria. Consistent with this observation, cell culture studies reveal that the mitochondrial isoform (DUT-M) is expressed constitutively, independent of cell cycle status. These data suggest that in normal tissues, nuclear staining with the DUT415 antibody represents the DUT-N isoform, whereas cytoplasmic staining represents the DUT-M isoform.
In colon cancer tumor specimens, expression of dUTPase was shown to be highly variable in both amount and intracellular localization. Patterns of dUTPase protein expression observed included exclusive nuclear, exclusive cytoplasmic, and combined nuclear and cytoplasmic staining. Thus, immunohistochemical detection of dUTPase in colon cancers provides distinct intracellular phenotypes of expression that may be of significant prognostic value.
To examine the association between dUTPase expression and response to 5-FU-based chemotherapy and overall survival, we initiated a retrospective study including tumor specimens from 20 patients who had received protracted infusion of 5-FU and leucovorin for treatment of metastatic colon cancer. Positive nuclear staining was found in 8 patients, whereas 12 lacked nuclear expression. Of the patients lacking nuclear dUTPase expression, 6 responded to 5-FU-based chemotherapy, 4 had stable disease, and 2 had progressive disease. Of the patients presenting positive nuclear dUTPase expression, 0 responded to chemotherapy, 1 had stable disease, and 7 had progressive disease (P = 0.005). The median survival for patients with tumors lacking nuclear staining was 8.5 months and 6.9 months for patients with tumors demonstrating positive nuclear dUTPase expression (P = 0.09). Time to progression was significantly longer for patients with tumors lacking nuclear staining (P = 0.017). Variable cytoplasmic dUTPase expression was observed in these tumors; however, there was no apparent association with clinical response or survival in this limited study. Nuclear dUTPase staining within these tumors was also associated with TS gene expression (P = 0.06).
This study demonstrates that low intratumoral levels of nuclear dUTPase protein expression is associated with response to 5-FU-based chemotherapy, greater time to progression, and greater overall survival in colorectal cancer. Conversely, high levels of nuclear dUTPase protein expression predict for tumor resistance to chemotherapy, shorter time to progression, and shorter overall survival. This report represents the first clinical study implicating dUTPase overexpression as a mechanism of resistance to TS inhibitor-based chemotherapy.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The methyl donor in this reaction, MTHF, is oxidized to dihydrofolate
so that the TS reaction constitutes a significant drain on cellular
tetrahydrofolate pools. The levels of MTHF are maintained during TMP
synthesis by the combined actions of DHFR and serine
hydroxymethyltransferase (Fig. 1A)
. Chemotherapeutic agents
such as 5-FU and FUdR block TMP biosynthesis by inhibiting TS directly.
Inhibitors of DHFR (e.g., methotrexate) indirectly block TMP
production by limiting the availability of MTHF (Fig. 1B)
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In virtually all known organisms, uracil is not a native component of DNA. However, uracil can arise in DNA either by the spontaneous deamination of cytosine residues or through dUTP utilization by DNA polymerases during replication (5 , 6) . Because cytosine deamination can lead to G:C to A:T transition mutations, the cell has evolved highly efficient mechanisms to facilitate the exclusion of uracil from DNA (7) . When uracil does occur in DNA, UDG initiates the base-excision repair pathway to remove and correct the misincorporated nucleotide. To prevent dUTP utilization during DNA replication, the enzyme dUTPase hydrolyzes dUTP to yield dUMP and PPi. This reaction effectively eliminates dUTP from the DNA biosynthetic pathway and also provides substrate (dUMP) for the de novo synthesis of thymidylate. Therefore, under normal cellular conditions, the maintenance of uracil-free DNA is achieved through the combined actions of dUTPase and UDG.
Although dUTP is a normal intermediate in thymidylate biosynthesis, its extensive accumulation and misincorporation into DNA is lethal in both prokaryotic and eukaryotic organisms (8 , 9) . The exact biochemical basis for uracil-DNA-mediated cell death has not been definitively proven; however, there is substantial evidence suggesting that UDG-initiated repair is a central component of this process. For example, inactivation of dUTPase in Escherichia coli results in the dramatic accumulation of dUTP pools leading to extensive uracil misincorporation during replication. Under conditions of elevated dUTP pools, the cell engages in repeated cycles of uracil misincorporation and UDG-mediated repair. This iterative process results in increased recombination, DNA strand breaks, and ultimately cell death (8) .
A similar phenomenon is thought to occur during inhibition of de
novo thymidylate metabolism by anticancer agents
(10, 11, 12, 13, 14, 15)
. Inhibition of the TS reaction leads to the
accumulation of cellular dUMP pools and, as a result of mono- and
diphosphate kinases, induces a dramatic increase in dUTP pools. Once
levels of dUTP accumulate beyond a threshold level, overwhelming
cellular dUTPase activity, the dUTP:TTP ratio increases. Under these
conditions, dUTP is misincorporated into replicating DNA, resulting in
uracil-DNA-mediated cytotoxicity (Fig. 1B)
. Implicit to this
model of cell killing is the central role of dUTPase. As the main
regulator of dUTP pools, the expression of dUTPase could have profound
effects on the utility of chemotherapeutics that inhibit thymidylate
biosynthesis. Normally, dUTPase mediates a protective role by limiting
the expansion of dUTP pools and countering the cytotoxic effect of
uracil misincorporation. According to this model, elevated levels of
dUTPase would prevent the accumulation of dUTP required for cell
killing. To test this hypothesis, Canman et al.
(16)
ectopically overexpressed the E. coli
dUTPase in a FUdR-sensitive human colorectal tumor cell line (HT29) and
measured the response to the TS inhibitor FUdR. The manipulated cell
lines (containing dUTPase activity 4–5-fold higher than controls) were
protected from FUdR-induced DNA strand breaks and showed an increased
viability over control cells (16)
. This study provided the
first evidence in human cells implicating dUTPase enzyme levels as an
important factor in determining the efficacy of TS inhibition. Although
the contribution of uracil-DNA-mediated cytotoxicity toward overall
cell death is likely to vary between different cell lines
(17)
, these data support the role of dUTP pool imbalance
and uracil misincorporation as a contributing mechanism of TS
inhibitor-based cytotoxicity.
Cancer of the gastrointestinal system is one of the leading causes of cancer death, and the fluoropyrimidines remain the chemotherapeutic agent of choice used to combat these diseases (18) . The difficulty in successfully treating patients with this class of drugs is the high incidence of resistance intrinsic to these cancers. Because resistance is a common phenomenon, understanding and predicting response and overall patient outcome is of great importance for the clinical evaluation and management of the cancer patient. Considerable effort has been directed toward identifying useful molecular markers that predict for patient response to chemotherapy and overall survival in gastrointestinal cancers. For example, recent advances have focused on measuring the intratumoral expression of molecular determinants of drug action (i.e., TS and TP) and mechanisms of response to DNA damage (e.g., p53; Refs. 19, 20, 21, 22, 23 ). However, there has been no clinical evaluation of potential predictive markers involved in dUTP metabolism. Considering the central role of dUTPase in uracil-DNA-mediated cytotoxicity, we conducted a retrospective study to evaluate the ability of dUTPase isoform expression to predict for response to 5-FU-based chemotherapy and overall patient survival in colorectal cancers.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture, Peripheral Blood Lymphocyte Preparation, and
Metabolic Labeling.
All cell lines were maintained in a humidified atmosphere of 5.5%
CO2 at 37°C. HeLa S3 cells (CCL 2.2) were
purchased from American Type Culture Collection and maintained in DMEM
supplemented with 5% FCS purchased from Life Technologies, Inc. Human
PBLs were prepared from venous whole blood with LeucoPREP cell
separation tubes (Becton Dickinson) using the manufacturer’s
recommendations. PBLs were cultured in RPMI 1640 supplemented with 10%
fetal bovine serum. PBLs were stimulated by the addition of PHA to a
final concentration of 15 µg/ml (Sigma). The onset of DNA replication
was monitored in stimulated PBLs by
[3H]thymidine incorporation. At 12-h intervals
after PHA stimulation, cells were labeled for 30 min with 10 µl of
[3H]thymidine (1 mCi/ml). The remainder of the
procedures was performed as described previously (26)
.
Cytospins and Immunocytochemistry.
Cultured cells were harvested at the appropriate times, washed in PBS,
counted, and resuspended in PBS at 3 x 105 cells/ml. Positively charged slides were
placed in cytocarriers (adapter 1024) for the IEC-Centra-8R centrifuge
(International Equipment Co.). One-ml aliquots of the cell suspension
were centrifuged at 400 x g for 5 min. Cells
were fixed in 4% paraformaldehyde in PBS (pH 7.2) for 30 min at room
temperature while the slides were still attached to the slide carrier.
Slides were removed from the carrier and washed five times for 2 min in
PBS (pH 7.0). Cells were then lightly digested with Proteinase K Dako
cat# 53020 diluted 1:500 with PBS for 15 min at room temperature. Cells
were washed again five times in PBS, and immunocytochemistry was
performed using the ABC-alkaline phosphatase protocols.
dUTPase-specific MAb (DUT415) was used at a concentration of 2 µg/ml
and incubated for 30 min at room temperature.
Immunohistochemistry.
Formalin-fixed, paraffin-embedded human tissue specimens were sectioned
at 4–5 µm and placed on Capillary Gap charged and precleaned glass
microscope slides (Ventana Medical Systems, Inc.). The tissue sections
were deparaffinized in xylene and rehydrated through graded alcohol
solutions. Prior to immunohistochemical labeling, epitope recovery was
performed by steam pretreatment (Ref. 27
and references
therein). Capillary Gap slide pairs were placed in a slide holder in 10
mM sodium citrate (pH 6.0). Water was brought to a boil in
a steamer (Handy Steamer Plus; Black and Decker), and the entire slide
holder was placed in the upper chamber of the steamer with the lid on
and steamed for 20 min. After steam pretreatment, the slides were
cooled for 10 min, rinsed in PBS for 5 min, and incubated in a 3%
hydrogen peroxide solution for 5 min to quench any endogenous
peroxidase activity. The tissues were rinsed again in PBS and placed in
normal goat serum (5% in PBS) for 5 min to reduce nonspecific
staining. The tissues were subsequently incubated with primary antibody
(e.g., DUT415) for 30 min at room temperature. After
incubation, the tissues were rinsed in PBS and incubated in
biotinylated goat-anti-mouse secondary antibody for 30 min (Vector
Laboratories). The slides were washed again in PBS to reduce
nonspecific staining of secondary antibody and incubated with
ABC-peroxidase for 30 min (Vector Elite). After a final rinse in PBS,
the tissues were incubated with DAB substrate (0.02% DAB, 0.005%
hydrogen peroxidase) for 15 min to develop the colorimetric reaction,
then counterstained in hematoxylin 100% (GILL 11 formula), and mounted
with glass coverslips using Permount.
Antibodies for Immunohistochemistry.
All antibodies were individually optimized for working dilutions. All
tissue samples were pretreated by steaming as described above for
antigen recovery. The DUT415 MAb was generated in our laboratory and
used at 2 µg/ml. Ki67 (MIB-1 clone) was purchased from Immunotech and
used at a dilution of 1:60. The mitochondrial marker 113-1 (Mito-M) was
purchased from BioGenex and used at a concentration of 1:100.
Patients.
Specimens from 20 patients with metastatic colon cancer were available
for analysis of dUTPase expression. These are a subset of 36 patients
who had received protracted infusion of 5-FU with leucovorin for
treatment of the metastatic disease and who had consented to have
biopsies for analysis of molecular markers. Tumor specimens from these
patients had been analyzed previously for TS gene
expression by reverse transcription-PCR (28)
.
Patients who experienced a complete response or partial response
(>50% reduction of measurable tumor) were classified as responders.
Survival was calculated as the time from start of treatment until death
of any cause or until last date known to be alive. Time to tumor
progression (>25% increase in tumor mass) was determined from
patients that had stable disease or responded to chemotherapy. Response
and survival information was available for all 20 patients included in
this study. See Table 1
for a summary of the demographic characteristics of these patients.
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RESULTS |
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In previous studies, we identified and characterized distinct
mitochondrial and nuclear isoforms of dUTPase in humans (DUT-M and
DUT-N, respectively) that have identical kinetic affinities for dUTP
(25)
. Detailed mass spectrometry-based analysis of native
dUTPase proteins revealed that the isoforms are largely identical,
differing only in a short region of their NH2
termini (25
, 31)
. Fig. 2A
summarizes these data in schematic format. The protein
sequences shown indicate native NH2-terminal
sequence and illustrate where the isoforms overlap in their primary
sequences. The identity of the amino acid sequence at the protein level
reflects the fact that the same nuclear gene encodes both isoforms,
with each isoform arising through the use of alternative 5' exons
(32)
. Additionally, the DUT-N isoform is phosphorylated on
Ser-11 in vivo (31)
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demonstrates that DUT415 is able to
immunoprecipitate both nuclear and mitochondrial isoforms of dUTPase.
The processed DUT-M protein (Mr
23,000) is readily resolved from the DUT-N protein, which has an
apparent molecular weight of Mr
22,000. These data demonstrate the cross-reactivity of DUT415, which is
likely attributable to the large region of homology common to these
isoproteins.
Expression of dUTPase Isoforms in PBLs.
Prior to this study, our knowledge of the expression of dUTPase
isoforms in human cells relied primarily on cell culture models. Using
normal human fibroblasts (34Lu cells), our laboratory has investigated
dUTPase isoform expression as a function of cellular proliferation
status. Data from these experiments demonstrate that DUT-N is regulated
in a growth-dependent manner, with expression correlating with the
onset of DNA replication at both the protein and mRNA levels. In
contrast, DUT-M is expressed in a constitutive fashion, mimicking the
pattern of mtDNA replication that occurs independently of nuclear DNA
replication (32
, 33)
. To establish the distribution of
dUTPase protein expression in human tissues, we have developed
methodologies to detect dUTPase by immunocyto- and immunohistochemistry
using DUT415. Initially, PBLs were examined as a primary cell culture
model. To provide a basis for the interpretation of isoform expression
in human tissues, immunoblot analysis and immunohistochemistry were
used in tandem to investigate the differential expression of dUTPase
isoforms after mitogenic stimulation of quiescent PBLs.
PBLs were isolated from venous whole blood and either maintained in
culture in a resting state or were stimulated by the addition of PHA.
Fig. 3A
illustrates a time-course experiment where protein extracts
from equivalent numbers of PBLs were fractionated by SDS-PAGE and
dUTPase was detected by Western blot analysis. Time points were taken
at 24 and 48 h after PHA stimulation, including negative controls.
Untreated PBLs express the DUT-M isoform at both 24- and 48-h time
points, whereas the DUT-N isoform is undetectable in these quiescent
cells. However, after PHA addition, DUT-N undergoes a dramatic
induction as PBLs are stimulated to proliferate. Analysis of DNA
replication by [3H] thymidine incorporation
indicates that PBLs begin DNA synthesis 
24 h after stimulation,
suggesting that DUT-N is up-regulated just prior to the onset of DNA
replication in these cells (data not shown). These data are in
agreement with the 34Lu fibroblast model, where DUT-M is constitutively
expressed and DUT-N expression is growth dependent (32)
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illustrates
dUTPase expression in PBLs that were either left untreated or
mitogenically stimulated with PHA for 48 h. The quiescent PBLs
exhibit no detectable staining using this methodology, suggesting that
the level of DUT-M protein in these cells is below the sensitivity of
this assay. In contrast, the PHA-stimulated cells demonstrate strong
nuclear expression of dUTPase, as indicated by the presence of red
chromogen. Preincubating the DUT415 antibody with a 5-fold excess of
recombinant antigen abolishes staining in the PHA treated cells (data
not shown). These observations are in agreement with the immunoblot
analysis, where DUT-N is dramatically up-regulated in stimulated cells.
These data also demonstrate the ability of the DUT415 antibody to
detect dUTPase protein in normal human cells by immunocytochemical
techniques.
Immunohistochemical Localization of dUTPase in Normal Human
Tissues.
To determine the expression of dUTPase in normal tissues, we developed
methodology to detect dUTPase in formalin-fixed and paraffin-embedded
human tissues by immunohistochemistry. In addition to dUTPase, the
proliferation marker Ki67 (MIB-1 clone) and the mitochondrial marker
[113-1 clone (Mito-M)] were used to stain serial sections of normal
tissue. These markers were used as controls in these experiments to
identify proliferating cells (Ki67) and cells that possess a rich
population of mitochondria (Mito-M; Refs. 34
and
35
). To establish the pattern of dUTPase expression
throughout the human body, we surveyed a panel of different normal
human tissues with the DUT415 antibody. Results from these experiments
reveal both nuclear and cytoplasmic staining by DUT415 (Fig. 4)
. An ABC-peroxidase technique was used in these experiments, and
protein expression is indicated by the presence of brown chromogen.
Nuclear dUTPase staining parallels Ki67 in highly proliferative cell
types in all cases throughout the human body. Examples include
proliferative cells of the immune system and replicating cells of
epithelia. Fig. 4A
illustrates nuclear staining by DUT415 in
palantine tonsil. The proliferating B cells of the germinal center and
stratum basale layer of the associated stratified squamous
epithelium stain in the nucleus with the DUT415 antibody. The
proliferation marker Ki67 demonstrates a similar pattern of expression,
as shown in Fig. 4B
. The nuclear staining of dUTPase in
proliferating cells of normal human tissues is consistent with the
studies of DUT-N expression in both 34Lu cells and PBLs, where DUT-N
protein is expressed in response to proliferation status. In addition,
DUT415 staining was consistent within and among individual tissue
samples. For example, reproducible staining patterns were observed in
10 different tonsil specimens, and repeated staining of the same
tissues yielded similar results.
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. In this image, the DUT415
MAb was used to stain the nonproliferative cells of the myocardium.
Although the nuclei are readily visible and lack detectable nuclear
dUTPase staining, these cells stain strongly in the cytoplasm with
DUT415. Staining of a serial section of myocardium with the Mito-M
polyclonal antibody reveals a similar staining pattern as DUT415 (Fig. 4D)
. In all cases tested, cytoplasmic expression of dUTPase
is correlated with a high mitochondrial content, as determined by
Mito-M immunoreactivity. Specific examples include the proximal
convoluted tubules of the kidney and thyroid follicular cells. The
cytoplasmic expression of dUTPase does not correlate with proliferation
status and is found in both replicating and nonreplicating cell types.
This observation is in agreement with dUTPase expression observed in
34Lu cells and PBLs in which expression of the DUT-M isoform occurs
independent of proliferation status. Together these data suggest that
the observed cytoplasmic staining in normal tissues represents the
mitochondrial isoform of dUTPase.
Fig. 4E
illustrates dUTPase expression in normal colonic
mucosa. Immunohistochemical staining of colon tumors will be described
in the next section; therefore, representative staining of DUT415 in
normal colon is included here as a reference. Nuclear staining is
observed in the replicating cells of the bottom half of the crypts of
Lieberkühn. There is also significant dUTPase staining in the
cytoplasm of these cells. Staining of colonic mucosa with Mito-M
antiserum demonstrates that these cells are rich in mitochondria,
suggesting that the cytoplasmic staining of dUTPase in these cells
represents DUT-M (data not shown).
To demonstrate the specificity of the DUT415 antibody in the
immunohistochemistry assay, we performed antigen competition analysis
on a serial section of normal colon tissue. DUT415 was preincubated
with 5-fold molar excess of purified recombinant dUTPase protein, and
immunohistochemistry was performed as described. Preincubation with
antigen completely abrogates staining with DUT415 in both the nucleus
and cytoplasm as illustrated in Fig. 4F
. Competition
experiments were also performed with several other tissues including,
tonsil, kidney, breast cancer, colon cancer, and melanoma. Identical to
normal colon tissue, staining with DUT415 was effectively competed with
recombinant antigen (data not shown).
The overall analysis of dUTPase expression in both cell culture and normal human tissues suggests that the pattern of intracellular distribution of dUTPase protein reflects the physiological status of the cell. Nuclear staining (DUT-N) is observed in proliferating cells, corresponding to an up-regulation of S phase-dependent gene products. In contrast, cytoplasmic staining (DUT-M) appears to reflect rich mitochondrial content within the cell that is indicative of a high metabolic status.
Immunohistochemical Detection of dUTPase in Neoplastic Tissues.
In an effort to establish the distribution and intracellular
localization of dUTPase expression in human cancers, we performed
immunohistochemistry on colon and gastric tumor samples. This analysis
was performed on formalin-fixed and paraffin-embedded tumor tissue
using the DUT415 antibody. Results from these experiments reveal that
dUTPase expression varies dramatically in different tumor specimens,
not only in the quantity of expression but also in the intracellular
localization. Our data show varied phenotypes of expression ranging
from exclusive cytoplasmic expression to exclusive nuclear expression.
In addition, there is evidence suggesting that dUTPase is aberrantly
expressed in certain tumors. Photomicrographs of dUTPase and Ki67
staining in two colon tumors are presented to illustrate examples of
the observed expression patterns.
In Fig. 5A
, dUTPase expression is highly expressed in both the
cytoplasm and nucleus within the tumor cells, while the surrounding
stromal cells exhibit very little dUTPase expression. A serial section
of the same tumor, stained with the proliferation marker Ki67,
demonstrates a characteristic nuclear staining pattern in the tumor
cells indicating a high proliferative status within this cancer (Fig. 5B)
.
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. This photomicrograph depicts the margin of the
tumor where both neoplastic tissue (left) and normal colonic
mucosa (right) can be observed. In this case, dUTPase
expression within the tumor cells is predominantly cytoplasmic (Fig. 5C)
. The colonic mucosa on the right side of this image
demonstrates characteristic nuclear dUTPase staining in the
replicating cells in the bottom half of the crypts of Lieberkühn,
providing an internal positive control for nuclear dUTPase
staining (see Fig. 4E
for comparison). Interestingly, a
serial section of the same tissue stained for Ki67 demonstrates strong
expression in the nucleus of both the tumor cells and the replicating
cells of the crypts (Fig. 5D)
. These data demonstrate that
nuclear dUTPase staining and Ki67 do not always correlate within tumor
tissues, suggesting that dUTPase isoforms may be aberrantly expressed
or incorrectly localized in certain tumors.
To further illustrate the variable intracellular distribution of
dUTPase in tumors, photomicrographs are presented in Fig. 6
of colon cancers stained with the DUT415 MAb. This panel of images
illustrates exclusive nuclear expression (Fig. 6, A and B)
, exclusive cytoplasmic expression (Fig. 6, C and D)
, and a combination of nuclear and cytoplasmic
expression (Fig. 6, E and F)
. It is significant
to note that the different patterns of dUTPase expression illustrated
are commonly observed in colon and gastric cancers. For example, a
study of dUTPase expression in 41 gastric tumors revealed that 27% had
nuclear and cytoplasmic staining, 12% had exclusive nuclear
expression, 39% had exclusive cytoplasmic expression, and 22% were
negative for dUTPase staining (data not shown). Additionally, the
phenotype of expression appears to be stable within a given tumor
specimen. A tumor sample exhibiting exclusive cytoplasmic expression
within one field of view retains this phenotype throughout the specimen
and tends not to display heterogeneous phenotypes. It is currently
unknown whether metastatic lesions exhibit different patterns of
expression relative to the primary tumor. The expression patterns of
dUTPase within colon and gastric cancers provide distinct and
recognizable phenotypes that together or independently may be of
significant prognostic value.
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Of the 20 samples tested, we found that 8 patients’ tumors contained
positive nuclear dUTPase staining (>10% positive nuclear expression).
There was a variable amount of cytoplasmic staining (ranging from
>75% to <5% tumor cells demonstrating cytoplasmic staining). None
of these patients responded (0%) to 5-FU-based chemotherapy. Only 1
patient had stable disease, and 7 patients demonstrated
progressive disease. From these 8 nuclear-positive patients, the
overall median TS gene expression was 5.35 x 10-3 (expressed as a ratio of TS:ß-actin). The
median survival in this group was 6.9 months, and time to tumor
progression was 2.7 months (Table 2)
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. Comparative analysis of these data indicates that nuclear dUTPase expression may be a statistically significant predictor of response to 5-FU-based chemotherapy (P = 0.005), time to progression (P = 0.017), and overall survival (P = 0.09) in metastatic colon cancer. High nuclear dUTPase expression is associated with poor response, shorter time to progression, and poorer survival. Conversely, low nuclear dUTPase staining is associated with response to chemotherapy, longer time to progression, and greater overall survival.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Investigation of the underlying molecular mechanisms of cell killing induced by inhibitors of thymidylate metabolism suggest that aberrant uracil-DNA metabolism plays a significant role in initiating DNA damage and cell death (as reviewed by Aherne and Brown 4 ). A growing body of evidence suggests that variation in the expression of dUTPase, the chief regulator of dUTP pools, may be a critical factor in determining the efficacy of a broad range of chemotherapeutic agents that target de novo thymidylate biosynthesis. The model of uracil-DNA-mediated cytotoxicity predicts that overexpression of dUTPase induces resistance to TS-directed chemotherapeutics by limiting drug-induced dUTP pool accumulation, thereby preventing uracil misincorporation into DNA. In contrast, low dUTPase expression would promote dUTP accumulation, thereby inducing greater sensitivity to these agents. To test this hypothesis and establish the value of dUTPase expression as a prognostic marker, we have developed methodology to measure dUTPase expression by immunohistochemistry using the MAb DUT415. This investigation is the first to characterize dUTPase isoform expression in normal and neoplastic tissues and to correlate nuclear dUTPase expression with response and survival in colorectal cancer.
The data presented in this study establish the expression patterns of dUTPase isoforms in normal tissues. Immunohistochemical analysis of dUTPase throughout the human body demonstrates that nuclear staining occurs in replicating cell types. Examples include the stratum basale of epithelial tissue, cells at the base of the crypts of Lieberkühn in the mucosa of the gastrointestinal tract, and proliferating lymphocytes. This observation is confirmed by identical staining with the proliferation marker Ki67. These data are consistent with the expression of DUT-N in cell culture models (e.g., PBLs and 34Lu human lung fibroblasts) where DUT-N protein and message are regulated in a growth-dependent manner (32) . Taken together, these data strongly suggest that nuclear staining by DUT415 in normal human tissues is indicative of DUT-N expression.
dUTPase staining is also observed in the cytoplasm of normal tissues
that have a high mitochondrial content. Examples include myocardial
tissue and the cells of the proximal convoluted tubules in the kidney.
Similar staining with the polyclonal antibody Mito-M that immunoreacts
with mitochondria supports this observation. The cytoplasmic expression
of dUTPase is independent of cellular proliferation status and is found
in both replicating and nonreplicating cell types. For example, the
replicating cells at the base of the crypts of Lieberkühn in the
colon demonstrate cytoplasmic dUTPase staining as well as the
nonreplicating cells of the myocardium (Fig. 4)
. These data are in
agreement with the constitutive expression of DUT-M observed in both
quiescent and replicating PBLs and 34Lu cells. We have also shown that
DUT-M mRNA levels remain constant in both quiescent and replicating
cells (32)
. Taken together, these data suggest that the
cytoplasmic staining detected in normal human tissues is indicative of
DUT-M expression. Thus, DUT415 may be a useful marker in normal tissues
to simultaneously identify replicating cell populations by nuclear
staining and mitochondrial content through cytoplasmic staining.
Immunohistochemical staining of dUTPase in colon cancers demonstrates that dUTPase expression varies dramatically in different tumor samples, both in magnitude and intracellular localization. Patterns of dUTPase expression range from exclusive nuclear or exclusive cytoplasmic staining to tandem expression of both. A recent report by Fleischmann et al. (40) confirms these data, where these authors observed variable levels of dUTPase expression in colorectal tumors. Thus, dUTPase expression among patient tumor samples is highly variable, exhibiting distinct and recognizable phenotypes of staining that may be of significant prognostic value.
To examine the association between dUTPase expression and response to
5-FU-based chemotherapy and overall survival, we initiated a
retrospective study including tumor specimens from 20 patients who had
received protracted infusion of 5-FU and LV for treatment of metastatic
colon cancer. In these colon cancer cases, nuclear dUTPase expression
was associated with resistance to 5-FU-based chemotherapy
(P = 0.005), shorter time to progression
(P = 0.017), and a shorter median survival
(P = 0.09). In contrast, tumors lacking
nuclear dUTPase expression were more responsive to chemotherapy and had
a longer time to progression and longer overall survival period (see
Table 2
). Significantly, no patients with nuclear positive dUTPase
expression responded to 5-FU-based chemotherapy, and all responders
demonstrated a nuclear negative phenotype. These data suggest that
elevated expression of dUTPase in the nucleus of tumor cells may
protect cells from the cytotoxic effect of uracil misincorporation
induced by inhibition of thymidylate metabolism. Variable cytoplasmic
expression of dUTPase was also observed in these tumor specimens;
however, no association between cytoplasmic staining and response or
survival was evident in this limited study.
The expression of nuclear dUTPase was also associated with TS gene expression within these tumors (P = 0.06). This observation may be expected because TS gene expression has already been shown to be a predictive marker of response to 5-FU-based chemotherapy in gastrointestinal cancers (19 , 21 , 28) . Larger studies will be required to assess whether tandem determination of dUTPase and TS will provide a more accurate prediction of tumor response to chemotherapy and overall survival compared with each marker independently.
The association between dUTPase expression and response to chemotherapy has several implications: (a) this study represents the first clinical data that support uracil-DNA-mediated cytotoxicity as a molecular mechanism of response to 5-FU-based chemotherapy; (b) the utility of dUTPase as a prognostic marker is not limited to 5-FU alone. Because the action of dUTPase is a downstream event of TS inhibition, dUTPase may be a useful marker for a number of commonly used chemotherapeutics that target either TS or DHFR; and (c) dUTPase has long been considered a viable chemotherapeutic target (reviewed by McIntosh and Haynes 41 ). Immunohistochemical detection of dUTPase isoforms will aid in the development and evaluation of dUTPase inhibitory compounds.
Variable cytoplasmic versus nuclear expression of dUTPase between tumor specimens raises a question about the consequences of TS inhibition on nuclear versus mtDNA. The existence of nuclear and mitochondrial isoforms of both dUTPase and uracil-DNA glycosylase suggest that the maintenance of uracil-free DNA is critical for the integrity of both nuclear DNA and mtDNA. Although there have been many studies of the effect of TS inhibition on nuclear DNA, little is known about the contribution of mtDNA degradation toward cell killing in tumors. Studies of the effects of thymidylate deprivation on lower eukaryotes and human cell lines suggest that there is a significant bias toward organelle DNA-specific mutagenesis and degradation (42, 43, 44, 45) . Studies of HeLa cells and normal human fibroblasts demonstrate that methotrexate and FUdR both induce mitochondria-specific mutagenesis and DNA degradation, an effect that is reversed by the addition of thymidine (42) . These data suggest that in certain cases, mtDNA degradation and not nuclear DNA degradation may be critical for response to chemotherapy.
Within this study, nuclear dUTPase expression was the important variable in predicting response and survival, suggesting that drug-induced nuclear DNA damage may be the ultimate mediator of cytotoxicity. Although this limited study failed to correlate cytoplasmic dUTPase expression with clinical response or survival, the striking variability of dUTPase expression in the cytoplasm of tumor cells derived from different patients suggest that overexpression of DUT-M may play a significant role in mediating protection of mtDNA from uracil-DNA-mediated degradation. The role of mtDNA degradation in overall fluoropyrimidine cytotoxicity and DUT-M-induced resistance awaits further investigation.
The results of this study demonstrate that DUT415 is capable of detecting human dUTPase isoforms in formalin-fixed, paraffin-embedded tissues by immunohistochemistry. In normal tissues, nuclear expression of dUTPase is observed in replicating cell types, whereas cytoplasmic expression is observed in mitochondria-rich tissues. In addition, we have measured dUTPase expression in colon cancer tumor specimens and correlated these data with clinical outcome and response to chemotherapy. Although additional studies will be needed to further establish the clinical utility of this marker, these data strongly implicate dUTPase as a significant predictor of survival and response to 5-FU-based chemotherapy in metastatic colon cancer. The ability to measure dUTPase expression in patient tumor specimens by immunohistochemistry will aid in determining the role of dUTPase in drug resistance and may lead to improved therapeutic strategies for patients treated with inhibitors of thymidylate metabolism.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Molecular Biology, University of Medicine
and Dentistry of New Jersey, School of Osteopathic Medicine, 2
Medical Center Drive, Stratford, NJ 08084. Phone: (856) 566-6043; Fax:
(856) 566-6232; E-mail: ladner{at}umdnj.edu 
2 The abbreviations used are: TS, thymidylate
synthase; MTHF, 5,10-methylenetetrahydrofolate; DHFR, dihydrofolate
reductase; 5-FU, 5-fluorouracil; FUdR, fluorodeoxyuridine; UDG,
uracil-DNA glycosylase; dUTPase, dUTP nucleotidohydrolase; MAb,
monoclonal antibody; PBL, peripheral blood lymphocyte; PHA,
phytohemagglutinin; DAB, diaminobenzidine; LV, leucovorin; mtDNA,
mitochondrial DNA; TP, thymidine phosphorylase.
Received 12/20/99. Accepted 4/28/00.
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