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Tumor Biology |
Division of Gastroenterology, Brigham and Women’s Hospital [R. J. G.], Cancer Biology, Dana-Farber Cancer Institute [R. J. G., H. L. F., Y. L., A. B. P.], Department of Pathology [Y. F.] and Division of Gastroenterology [L. M. S.], Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(troglitazone and BRL46593) and of
retinoid X receptor (LG268), and histone deacetylase inhibitors
(trichostatin A, suberoylanilide hydroxamic acid, and tributyrin). A
synergistic induction of Drg-1 expression was seen with
the combination of tributyrin and a low dose of 5'-aza-2'-dexoycytidine
(100 nM), an inhibitor of DNA methylation. Functional
studies revealed that overexpression of Drg-1 in
metastatic colon cancer cells reduced in vitro invasion
through Matrigel and suppressed in vivo liver metastases
in nude mice. We propose that Drg-1 suppresses colon
cancer metastasis by inducing colon cancer cell differentiation and
partially reversing the metastatic phenotype. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To further define gene expression changes in metastatic colon
cancer, we used differential display and identified 19 genes that are
expressed differentially between primary and metastatic colon cancer.
One of these genes is identical to a gene identified previously named
Drg3
-1, which was found to be down-regulated in colonic adenomas
and primary colon cancer (3)
. The expression of this gene
was also found to be regulated by homocysteine, testosterone, and
Ni2+ in different cell types (4, 5, 6)
.
However, the function of this gene remains unknown. We now report that
Drg-1 is further down-regulated in metastatic colon cancer
cells to levels that are nearly undetectable when compared with the
primary colon cancer counterparts. We have further demonstrated that
stable transfection of a metastatic colon cancer cell line SW620 with
Drg-1 cDNA induced morphological changes indicative of
differentiation, up-regulated the expression of several colonic
epithelial cell differentiation markers, and reduced in
vitro invasion through Matrigel and in vivo liver
metastasis in nude mice. In mechanistic studies, we found that the
expression of Drg-1 was controlled by several
differentiation reagents, such as ligands of PPAR and the retinoid X
receptor, as well as by reagents affecting DNA methylation and histone
acetylation. These data suggest that Drg-1 may suppress
colon cancer metastasis by inducing cell differentiation and reversing
the metastatic phenotype.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemical and Biological Reagents.
Aza, all-trans retinoic acid, tributyrin, and TSA were
purchased from Sigma Chemical Co. (St. Louis, MO). LG268, a retinoid X
receptor selective ligand, was a gift from Richard A. Heyman (Ligand
Pharmaceuticals, San Diego, CA). Troglitazone and BRL49653, ligands of
PPAR, were gifts from Dr. Bruce M. Spiegelman at Dana-Farber Cancer
Institute. SAHA, a second-generation hybrid polar cytodifferentiation
agent shown to inhibit histone deacetylase and induce terminal
differentiation in transformed cells (7)
, was a gift from
Dr. Paul Marks (Sloan-Kettering Cancer Center, New York, NY).
DD.
SW480 and SW620 cell lines were both derived from the same colon
cancer patient. SW480 was established from a primary colon cancer
lesion, and SW620 was from a lymph node metastasis (8)
. To
ensure that the observed differences were not an artifact of long-term
cell culture, we also studied freshly isolated primary colon cancer
tissue and lymph node metastasis from a single patient. DD was
performed with a DD kit purchased from GenHunter Corp. (Nashville, TN),
according to the manufacturer’s protocol (9)
. The anchor
and arbitrary primers that led to detection of Drg-1 were
5'-AAGCTTTTTTTTTTTG-3' and 5'-AAGCTTTGGTCAG-3'. Band isolation and
direct sequencing of the DD band were performed as described
(9)
.
RNA Isolations and Northern Blot Analysis.
RNA from colon cancer cells was isolated with TRIzol reagent (Life
Technologies, Inc., Rockville, MD), according to the manufacturer’s
protocol. RNA from colon cancer tissues was isolated by the guanidinium
thiocyanate/CsCl method, as described (10)
. A
multiple-tissue dot blot was obtained from Clontech (Palo Alto,
California). Northern and dot blot analysis were performed as described
(10)
, except ExpressHyb hybridization solution from
Clontech was used. Nucleotides 4–337 of Drg-1 were
32P-labeled with a random labeling kit
(Boehringer Mannheim, Indianapolis, IN) and used as a probe for Drg-1.
Probes for E-cadherin and CEA were produced as described (11
, 12)
. The probe for PPAR was a gift from Bruce M. Spiegelman
at Dana-Farber Cancer Institute (13)
. Membranes were
hybridized in ExpressHyb hybridization solution (Clontech) with
32P-labeled probe, washed, and exposed to a
PhosphorImager (Bio-Rad Laboratories, Richmond, CA) or X-ray
films. The signal intensities were quantified with Imagequant software
(Bio-Rad Laboratories) and normalized to 28S RNA expression.
Generation of Drg-1 Stable Transfectants.
The coding region of Drg-1 (nucleotides 110-1346) was
cloned from a human normal prostate cDNA library (Clontech) by PCR with
Advantage cDNA Polymerase Mix from Clontech. The coding region of
Drg-1 cDNA was inserted in-frame into the pcDNA3.1 vector,
which contains the cytomegalovirus enhancer-promoter (Invitrogen Corp.,
Carlsbad, CA). The cDNA was then fully sequenced to ensure that no
mutations were introduced during the PCR amplification. SW620 colon
cancer cells were seeded in 0.6-cm dishes at 5 x 105 cells/dish and transfected with a pcDNA3.1
vector containing Drg-1 cDNA or with an empty vector as
control using Superfect (Qiagen, Inc., Valencia, CA), according to the
manufacturer’s protocol. After culturing in medium containing 0.8
mg/ml of G418 (LifeTechnologies, Inc.) for 
2 weeks, individual
clones were isolated using cloning cylinders. The cell clones that
expressed the 1.2-kb Drg-1 cDNA coding region (as confirmed
by Northern blot) were maintained in medium containing 0.2 mg/ml of
G418 and used for further investigation.
Alkaline Phosphatase Assays.
Alkaline phosphatase assays were performed as described
(14)
. Control cells and different Drg-1
expressed cell clones were cultured for 48 h to half confluence
and lysed. Alkaline phosphatase activity in cell lysates was determined
with p-nitrophenyl phosphate disodium hexahydrate (Sigma
104) as a substrate. Synthetic alkaline phosphatase (Life Technologies)
was used to construct a standard dilution curve. Each assay was
performed in triplicate. The means ± SE from two
separate experiments are presented.
In Vitro Matrigel Invasion Assay and in
Vivo Nude Mice Studies.
In vitro Matrigel invasion assays were performed as
described using 6.5-mm transwell chambers (8-µm pore size; Costar).
The transwell filters were coated with 5 µg of Matrigel
(15)
. SW620 cells (1 x 105) overexpressing Drg-1 or vector
control cells were cultured in the upper chamber, and conditioned NIH
3T3 medium was added to the bottom chamber. After 72 h, the cells
were fixed and stained, and the number of cells that invaded through
the Matrigel was quantified as described (15)
.
Animal protocols were approved by the Institutional Animal Care and Use Committee at the Dana-Farber Cancer Institute and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Female BALB/c athymic nude mice (National Cancer Institute Frederick Cancer Research Facility, Rockville, MD), 8 weeks of age, were anesthetized with methoxyfluorane by inhalation, and a small abdominal incision was made under sterile conditions. Viable tumor cells (5 x 105) in 0.02 ml of serum-free medium were injected into the spleen by means of a sterile tuberculin syringe and a 30-gauge needle. During the injection, care was taken to maintain uniform cell suspensions and to avoid injecting clumped cells. The injection site was then dabbed gently with sterile gauze dampened with 95% ethanol to kill tumor cells that may have escaped. The abdomen was closed with a metal surgical clip, and the animals were returned to their cages. After 8 weeks, the animals were euthanized. The spleen and liver were weighed, as well as examined for splenic "primary" tumor and liver metastases by visual inspection. Metastases were confirmed with histological studies as described (16) .
Statistical Analysis.
Statistical computations were performed using the statistical analysis
systems statXact (Statistics Unlimited, Savanna, GA). For the
statistical analysis of the difference between control and transfected
cells in the Matrigel assay, ANOVA with the Tukey correction for
multiple comparisons to provide a nominal significance level (
) of
0.05 was used. For the statistical analysis of the difference between
control and transfected cells in the animal study, the Fisher exact
test with an appropriate cutoff P of <0.05 was used.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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illustrates a typical example of DD (Fig. 1A)
and a Northern
blot (Fig. 1B)
, which confirms the existence of 3.0-kb mRNA
that is expressed in the SW480 primary colon cancer cells and the
primary colon cancer tissues but not in their metastatic counterparts.
To determine the identity of this gene, the DD band (Fig. 1A)
was extracted, reamplified, and sequenced. A BLAST
computer database search found that this cDNA was 99% homologous to
the 3' untranslated region of Drg-1 (3)
, thus
revealing its identity.
|
. The role of DNA methylation on the expression of this
gene will be discussed in detail below. To extend these findings, we
studied a total of eight colon cancer cell lines and 10 human colon
cancer specimens (5 primary tumors paired with 5 metastatic colon
cancer lesions; Fig. 2
). Of the four cell lines with the lowest level
of Drg-1 expression (Fig. 2A)
, three (SW620,
LoVo, and Colo205) were derived from metastatic lesions of colon
cancer, and the fourth, SW48, was derived from a poorly differentiated
primary colon cancer. In contrast, the other four cell lines (SW480,
DLD-1, HCT116, and CaCO2) were derived from
primary colon cancer lesions (20, 21, 22)
. Similarly, the
expression of this gene was also found to be substantially
down-regulated in two and completely undetectable in three metastatic
lesions (Fig. 2B)
when compared with the paired primary
colon cancer lesions. In preliminary Northern blot studies of 36
clinical specimens of breast cancers (data not shown), the level of
Drg-1 expression did not differ appreciably between primary
breast cancers and metastatic lesions. These data suggest that
Drg-1 may be specific for colon cancers.
|
, the expression of Drg-1 was found in all tissues tested
with a slightly higher expression level in the brain, prostate, and
adult and fetal kidney, as well as placenta. The same blot was stripped
and reprobed with ubiquitin to ensure equal loading (data not shown).
The absence of signals in bacterial or yeast cDNA samples demonstrated
the specificity of the probing. The presence of signals in human DNA
(Fig. 3
, right lower corner) suggests that Drg-1
is highly abundant in human tissue or belongs to a multigene family.
The ubiquitous expression of Drg-1 suggests that this gene
may function as a housekeeping gene.
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Although the growth rate of the transfected cells was very
similar to that of the neo controls and parental
nontransfected cells (data not shown), distinct morphological changes
were seen in the Drg-1-transfected cells (Fig. 4)
. The transfected cells were larger, flatter, and spindle shaped (Fig. 4B)
, in contrast to the smaller, more round neo
control cells (Fig. 4A)
. These morphological changes were
consistently observed in multiple transfected clones expressing
Drg-1 but not in those clones that did not express
Drg-1. Similar morphological changes were seen when
differentiation was induced in parental SW620 by known differentiation
reagents such as tributyrin, a prodrug of butyrate (Fig. 4C)
, LG268, a ligand of RXR (Fig. 4D)
, and
all-trans retinoic acid, a ligand of retinoic acid receptor
(not shown). These findings suggest that the expression of
Drg-1 induces changes characteristic of cell differentiation
in colon cancer cells.
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, the activity of alkaline phosphatase was 2–3-fold higher
in all five transfected cell clones (620/T) compared with the
neo control cells (620/V). Similarly, the expression of
E-cadherin and CEA was also up-regulated in all transfected cell
clones, although the levels of expression varied among individual
clones (Fig. 5B)
. Together, these findings suggest that the
expression of Drg-1 induces colon cancer cell
differentiation.
|
, RXR, DNA Methylation, and Histone Acetylation.
and 6B
, treatment with Aza, an inhibitor of DNA methylation and a
known differentiation inducer (19)
, partially up-regulated
the expression of Drg-1 in all colon cancer cell lines
tested, suggesting that the expression of Drg-1 is
controlled by DNA methylation. PPAR is a ligand-inducible
transcription factor known to control differentiation of a variety of
cells including adipocytes and colonic epithelial cells
(13)
. To investigate the potential functional association
between the PPAR and Drg-1, we first studied the
expression of PPAR in Drg-1-transfected and
neo control SW620 cells. Northern blot studies with a probe
specific for PPAR revealed that its expression level in both
transfected and neo control SW620 cells is nearly identical
(data not shown), suggesting that the expression of PPAR is
independent of Drg-1. On the other hand, the expression of
Drg-1 was up-regulated by troglitazone (Fig. 6A)
and BRL46593 (not shown), two synthetic ligands of PPAR
(13)
, suggesting that Drg-1 is actually
downstream of PPAR. In addition, LG268, a synthetic ligand specific
for RXR (23)
also up-regulated Drg-1 (Fig. 6A)
. A combination of troglitazone and LG268 induced
Drg-1 expression by 10-fold, suggesting a possible
synergistic effect from these two drugs. Moreover, the expression of
Drg-1 was also markedly up-regulated by two histone
deacetylase inhibitors, TSA and SAHA (7
, 24
, 25) . The
effect of another histone deacetylase inhibitor, tributyrin (a prodrug
of butyrate), was only seen after 5 days of treatment (Fig. 6B)
. However, when cells were treated with tributyrin plus a
low dose of Aza (0.1 µM), a marked increase in
Drg-1 expression was seen (Fig. 6B)
, suggesting a
synergistic effect from these two drugs. Together, these data suggest
that Drg-1 may be a downstream element of the PPAR transcriptional
pathway and is controlled by both DNA methylation and histone
acetylation, two global mechanisms of gene regulation
(26)
. We suggest that Drg-1 may suppress colon
cancer metastasis by inducing cell differentiation and reversing the
metastatic phenotype.
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, the metastatic colon cancer cell line (SW620) migrated through the
Matrigel at levels about five times greater than the primary colon
cancer cell line (SW480), in agreement with data published previously
(27)
. Expression of Drg-1 cDNA in SW620 cells
(T1 and T5) reduced Matrigel invasion by up to 70%
(P < 0.0001). Expression of the
neo control vector had little effect on Matrigel invasion.
These data suggest that the overexpression of Drg-1 inhibits
the in vitro invasion ability of metastatic colon cancer
cells.
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, 12 of 14 mice injected with neo control cells and 13 of 15
mice injected with transfected cells developed "primary" tumors in
the spleen. The tumor burdens of the splenic primaries, as judged by
their weights, were very similar between the transfected and the
neo control group (data not shown). However, in the mice
that developed "primary" tumors, 9 of 12 mice (75%) developed
liver metastases in the neo control group, whereas only 3 of 13 mice
(23%) had liver metastases in the Drg-1-transfected group.
Statistical analysis using the Fisher exact test revealed a
P of 0.0169, suggesting that the differences in liver
metastases observed between the control and transfected groups are
unlikely attributable to chance alone. Therefore, these findings
suggest that Drg-1 may function as a suppressor of colon
cancer metastasis. These results also indicate that Drg-1
did not alter the ability of cancer cells to form primary tumors.
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|
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using DD, we have identified 19 genes expressed differentially between primary and metastatic colon cancer. One of these genes, Drg-1, was found to be down-regulated in metastatic colon cancer tissues and cell lines. Overexpression of Drg-1 induced morphological and molecular changes consistent with colon cancer cell differentiation and suppressed in vitro invasion and in vivo liver metastases in nude mice. Drg-1 was initially identified by comparing gene expressions between undifferentiated and well-differentiated HT-29 colon cancer cell lines (3) . Simultaneously, others found that Drg-1 was regulated by homocysteine in endothelial cells (4) , testosterone in T-cell hybridoma 312.13 cells (5) , and Ni2+ in human and rodent cell lines (6) , implying that Drg-1 may be a housekeeping gene (4) . In fact, a GenBank search revealed that the murine homologue of Drg-1 (named Ndr1, accession no. U60593) is a downstream target of N-myc, first suggesting that Drg-1 may be involved in cell growth and differentiation.
In the present study, we demonstrated that overexpression of Drg-1 induced distinct morphological changes similar to those observed during colonic epithelial cell differentiation. These morphological changes are associated with increased expression of several cell differentiation markers, suggesting that Drg-1 may function as a promoter of colonic epithelial cell differentiation. Moreover, the expression of Drg-1 is controlled by several known cell differentiation reagents. These results further support the notion that Drg-1 may be a key element in colonic epithelial cell differentiation. In addition, we have demonstrated that overexpression of Drg-1 in metastatic colon cancer cells suppress liver metastases in nude mice but do not alter the ability to form primary tumors. Together, these results suggest that induction of Drg-1 expression is capable of overriding the existing genetic defects and partially reversing the metastatic phenotype.
Our results indicate that the expression of Drg-1 is controlled by at least three mechanisms:
(a) PPAR/RXR transcriptional factor pathway. PPAR is a
member of the nuclear receptor superfamily that includes receptors for
steroids, thyroid hormone, vitamin D, and retinoic acid
(31)
. Ligands of PPAR include polyunsaturated fatty
acids such as linoleic, PGJ2, and the synthetic antidiabetic
thiazolidinedione drugs, troglitazone and BRL 49653 (32
, 33)
. Although dimerizing with the RXR receptor, PPAR
functions as a transcription factor, controlling differentiation of a
variety of cells including adipocytes and colonic epithelial cells
(34
, 35)
. Therefore, the finding that the ligands of
PPAR and RXR activate Drg-1 suggests that
Drg-1 may be a downstream target of the PPAR/RXR
differentiation pathway.
(b) DNA methylation pathway. It is well known that methylation of CpG islands in promoter sequences suppresses gene expression. Inhibition of DNA methylation by Aza induces differentiation of many cell types including colon cancer cells (36 , 37) . The 5' end of the Drg-1 cDNA contains multiple CpG sites, which first suggested that Drg-1 may be controlled by DNA methylation. We have now cloned and sequenced 800 bp of the Drg-1 promoter region (data not shown). Analysis of this sequence reveals that there are multiple CpG sites, sufficient to comprise a CpG island (17) . Additional studies to compare the promoter activity of Drg-1 with its methylation status will determine the role of DNA methylation in the regulation of Drg-1 expression. Our data also indicate that the expression of Drg-1 is only partially regulated by DNA methylation, implying that other mechanisms are involved in the down-regulation of Drg-1 in metastatic colon cancer cells.
(c) Histone deacetylation pathway. Inhibition of histone deacetylase by reagents such as butyrate and trichostatin has been shown to induce differentiation of many different cell types (7 , 38) . Our data demonstrate that inhibition of histone deacetylase induces the expression of Drg-1.
The synergistic effect of Aza and tributyrin on Drg-1
expression is of interest. The similar effect between an inhibitor of
DNA methylation (Aza) and an inhibitor of histone deacetylation (TSA)
also resulted in reexpression of genes such as p16 and
MLH1, which are silenced in cancers (39)
.
Together, these findings suggest that DNA methylation and histone
acetylation, two key processes controlling gene regulation, cell
growth, and cell differentiation, may be functionally linked. Because
the degree of histone acetylation depends on the balance of acetylation
and deacetylation, demethylated DNA may be a prerequisite condition for
recruitment of acetyltransferase enzyme and histone acetylation. In
this regard, recent studies by two independent groups have reported
that MeCP2, a methyl-CpG-binding protein, interacts with histone
deacetylase and induces transcriptional silencing by inducing histone
deacetylation (40
, 41)
. Additional studies of the
regulatory mechanism of Drg-1 may provide insight about the
interaction among transcription factors such as PPAR as
gene-specific regulatory mechanisms, as well as more global regulations
such as DNA methylation and histone acetylation (42)
.
Cytodifferentiation therapies have been used in the treatment of human malignancies for decades (43) . The fundamental mechanism of this approach is to "push" poorly differentiated tumor cells back into a genetic pathway of maturation/differentiation and, therefore, to reverse the malignant phenotype of tumor cells. The execution of this therapy, however, is only possible with an understanding of the relevant molecules that control cell differentiation and a realistic approach to manipulate the function of such molecules. Results from the present studies suggest that Drg-1 may be one of the molecules that plays a key role in controlling colonic epithelial cell differentiation. The fact that overexpression of Drg-1 induced expression of E-cadherin and two other cell differentiation markers, as well as induced morphological changes typical of differentiated cells, strongly suggests that SW620 metastatic colon cancer cells were "pushed" back into the differentiation pathway. Alterations of cell surface molecules, such as E-cadherin and possibly other cell surface molecules, may change the adhesion properties of cancer cells and result in the suppression of their in vitro and in vivo invasion capabilities (44 , 45) .
From a clinical point of view, decreased expression of
Drg-1 in colon cancer cells may be used as a potential
genetic marker to predict early metastasis. This can be achieved by
analyzing the expression of Drg-1 in primary colon cancer
using in situ hybridization or immunochemical studies,
techniques that allow the identification of Drg-1 expression
in individual colon cancer cells as compared with normal adjacent
tissue. Moreover, ligands of PPAR, RXR, or histone deacetylase
inhibitors might be used as pharmacological agents to induce the
expression of Drg-1 and thereby possibly reduce the invasion
and metastatic abilities of colon cancer cells. Specifically targeting
and manipulating the function of Drg-1 may offer a novel
approach to the differentiation therapy of colon cancer.
|
ACKNOWLEDGMENTS |
|---|
, Dr. Richard A. Heyman for
RXR ligand, Dr. Paul Marks for cytodifferentiation agent SAHA, and Dr.
Peter Choo from the Channing Laboratory, Brigham and Women’s Hospital,
for assistance with statistical analysis. |
FOOTNOTES |
|---|
1 Supported by Grant R0-1 CA61253 from the NIH (to
A. B. P.). 
2 To whom requests for reprints should be
addressed, at Dana-Farber Cancer Institute, D602, 44 Binney Street,
Boston, MA 02115. Phone: (617) 632-4683; Fax: (617) 632-4680.
3 The abbreviations used are: Drg,
differentiation-related gene; PPAR, peroxisome proliferator-activated
receptor ; Aza, 5'-aza-2'-deoxycytidine; TSA, trichostatin A; SAHA,
N-hydroxy-N'-phenyl-octane-1,8-diotic
acid diamide suberoylanilide hydroxamic acid; DD, differential display;
CEA, carcinoembryonic antigen; RXR, retinoid X receptor.
Received 7/23/99. Accepted 12/ 2/99.
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Z. Kovacevic and D. R. Richardson The metastasis suppressor, Ndrg-1: a new ally in the fight against cancer Carcinogenesis, December 1, 2006; 27(12): 2355 - 2366. [Abstract] [Full Text] [PDF]
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Y. Maruyama, M. Ono, A. Kawahara, T. Yokoyama, Y. Basaki, M. Kage, S. Aoyagi, H. Kinoshita, and M. Kuwano Tumor Growth Suppression in Pancreatic Cancer by a Putative Metastasis Suppressor Gene Cap43/NDRG1/Drg-1 through Modulation of Angiogenesis. Cancer Res., June 15, 2006; 66(12): 6233 - 6242. [Abstract] [Full Text] [PDF]
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 A. Fotovati, T. Fujii, M. Yamaguchi, M. Kage, K. Shirouzu, S. Oie, Y. Basaki, M. Ono, H. Yamana, and M. Kuwano 17{beta}-Estradiol Induces Down-Regulation of Cap43/NDRG1/Drg-1, a Putative Differentiation-Related and Metastasis Suppressor Gene, in Human Breast Cancer Cells. Clin. Cancer Res., May 15, 2006; 12(10): 3010 - 3018. [Abstract] [Full Text] [PDF]
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 B. Chen, D. M. Nelson, and Y. Sadovsky N-Myc Down-regulated Gene 1 Modulates the Response of Term Human Trophoblasts to Hypoxic Injury J. Biol. Chem., February 3, 2006; 281(5): 2764 - 2772. [Abstract] [Full Text] [PDF]
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 J. Su, L. He, N. Zhang, and P. C. Ho Evaluation of Tributyrin Lipid Emulsion with Affinity to Low-Density Lipoprotein: Pharmacokinetics in Adult Male Wistar Rats and Cellular Activity on Caco-2 and HepG2 Cell Lines J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 62 - 70. [Abstract] [Full Text] [PDF]
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Z. Dong, R. J. Arnold, Y. Yang, M. H. Park, P. Hrncirova, Y. Mechref, M. V. Novotny, and J.-T. Zhang Modulation of Differentiation-related Gene 1 Expression by Cell Cycle Blocker Mimosine, Revealed by Proteomic Analysis Mol. Cell. Proteomics, July 1, 2005; 4(7): 993 - 1001. [Abstract] [Full Text] [PDF]
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M. A. Shah, J. Kortmansky, M. Motwani, M. Drobnjak, M. Gonen, S. Yi, A. Weyerbacher, C. Cordon-Cardo, R. Lefkowitz, B. Brenner, et al. A Phase I Clinical Trial of the Sequential Combination of Irinotecan Followed by Flavopiridol Clin. Cancer Res., May 15, 2005; 11(10): 3836 - 3845. [Abstract] [Full Text] [PDF]
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M. A. Shah, N. Kemeny, A. Hummer, M. Drobnjak, M. Motwani, C. Cordon-Cardo, M. Gonen, and G. K. Schwartz Drg1 Expression in 131 Colorectal Liver Metastases: Correlation with Clinical Variables and Patient Outcomes Clin. Cancer Res., May 1, 2005; 11(9): 3296 - 3302. [Abstract] [Full Text] [PDF]
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 N Nathoo, A Chahlavi, G H Barnett, and S A Toms Pathobiology of brain metastases J. Clin. Pathol., March 1, 2005; 58(3): 237 - 242. [Abstract] [Full Text] [PDF]
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S. Stein, E. K. Thomas, B. Herzog, M. D. Westfall, J. V. Rocheleau, R. S. Jackson II, M. Wang, and P. Liang NDRG1 Is Necessary for p53-dependent Apoptosis J. Biol. Chem., November 19, 2004; 279(47): 48930 - 48940. [Abstract] [Full Text] [PDF]
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S. Bandyopadhyay, S. K. Pai, S. Hirota, S. Hosobe, T. Tsukada, K. Miura, Y. Takano, K. Saito, T. Commes, D. Piquemal, et al. PTEN Up-Regulates the Tumor Metastasis Suppressor Gene Drg-1 in Prostate and Breast Cancer Cancer Res., November 1, 2004; 64(21): 7655 - 7660. [Abstract] [Full Text] [PDF]
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K.-t. Kim, P. P. Ongusaha, Y.-K. Hong, S. K. Kurdistani, M. Nakamura, K. P. Lu, and S. W. Lee Function of Drg1/Rit42 in p53-dependent Mitotic Spindle Checkpoint J. Biol. Chem., September 10, 2004; 279(37): 38597 - 38602. [Abstract] [Full Text] [PDF]
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 T. Okuda, Y. Higashi, K. Kokame, C. Tanaka, H. Kondoh, and T. Miyata Ndrg1-Deficient Mice Exhibit a Progressive Demyelinating Disorder of Peripheral Nerves Mol. Cell. Biol., May 1, 2004; 24(9): 3949 - 3956. [Abstract] [Full Text] [PDF]
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J. Li, G. Davidson, Y. Huang, B.-H. Jiang, X. Shi, M. Costa, and C. Huang Nickel Compounds Act through Phosphatidylinositol-3-kinase/Akt-Dependent, p70S6k-Independent Pathway to Induce Hypoxia Inducible Factor Transactivation and Cap43 Expression in Mouse Epidermal Cl41 Cells Cancer Res., January 1, 2004; 64(1): 94 - 101. [Abstract] [Full Text] [PDF]
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A. Kehlen, U. Lendeckel, H. Dralle, J. Langner, and C. Hoang-Vu Biological Significance of Aminopeptidase N/CD13 in Thyroid Carcinomas Cancer Res., December 1, 2003; 63(23): 8500 - 8506. [Abstract] [Full Text] [PDF]
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Y. Wakisaka, A. Furuta, K. Masuda, W. Morikawa, M. Kuwano, and T. Iwaki Cellular Distribution of NDRG1 Protein in the Rat Kidney and Brain During Normal Postnatal Development J. Histochem. Cytochem., November 1, 2003; 51(11): 1515 - 1525. [Abstract] [Full Text] [PDF]
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S. Bandyopadhyay, S. K. Pai, S. C. Gross, S. Hirota, S. Hosobe, K. Miura, K. Saito, T. Commes, S. Hayashi, M. Watabe, et al. The Drg-1 Gene Suppresses Tumor Metastasis in Prostate Cancer Cancer Res., April 15, 2003; 63(8): 1731 - 1736. [Abstract] [Full Text] [PDF]
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S. Boulkroun, M. Fay, M.-C. Zennaro, B. Escoubet, F. Jaisser, M. Blot-Chabaud, N. Farman, and N. Courtois-Coutry Characterization of Rat NDRG2 (N-Myc Downstream Regulated Gene 2), a Novel Early Mineralocorticoid-specific Induced Gene J. Biol. Chem., August 23, 2002; 277(35): 31506 - 31515. [Abstract] [Full Text] [PDF]
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M. Motwani, F. M. Sirotnak, Y. She, T. Commes, and G. K. Schwartz Drg1, a Novel Target for Modulating Sensitivity to CPT-11 in Colon Cancer Cells Cancer Res., July 15, 2002; 62(14): 3950 - 3955. [Abstract] [Full Text] [PDF]
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 A Saito, G Fujii, Y Sato, M Gotoh, M Sakamoto, G Toda, and S Hirohashi Detection of genes expressed in primary colon cancers by in situ hybridisation: overexpression of RACK 1 Mol. Pathol., February 1, 2002; 55(1): 34 - 39. [Abstract] [Full Text] [PDF]
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A. Nishie, K. Masuda, M. Otsubo, T. Migita, M. Tsuneyoshi, K. Kohno, T. Shuin, S. Naito, M. Ono, and M. Kuwano High Expression of the Cap43 Gene in Infiltrating Macrophages of Human Renal Cell Carcinomas Clin. Cancer Res., July 1, 2001; 7(7): 2145 - 2151. [Abstract] [Full Text] [PDF]
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M. Matsushima-Nishiu, M. Unoki, K. Ono, T. Tsunoda, T. Minaguchi, H. Kuramoto, M. Nishida, T. Satoh, T. Tanaka, and Y. Nakamura Growth and Gene Expression Profile Analyses of Endometrial Cancer Cells Expressing Exogenous PTEN Cancer Res., May 1, 2001; 61(9): 3741 - 3749. [Abstract] [Full Text]
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