Proceedings
of the
American Association for Cancer Research
The
following abstracts were presented at the 93rd annual
meeting of the American Association for Cancer Research
(AACR). This meeting
was held in San Francisco from April 6-10, 2002.
Each
abstract was developed by a member(s) of our distinguished
faculty at NewYork Presbyterian Hospital or affiliated
medical schools, including the College of Physicians and
Surgeons of Columbia University and Weill Medical College
of Cornell University. In some instances, the work was
carried out in collaboration with researchers from other
institutions. While these abstracts span across cancer
research activities at New York Presbyterian Hospital,
the majority focus on the etiology and prevention of cancer.
To
go directly to abstracts in a particular area of interest,
click onto the following links:
Lab Research
Human Research
LAB
RESEARCH
A
mouse model of oral cavity carcinogenesis
Xiao-Han Tang, Debbie Bemis, Beatrice Knudsen, Lorraine
Gudas, Department of Pharmacology, Weill Medical College,
Cornell University, New York, NY; Department of Pathology,
Weill Medical College, Cornell University, New York, NY.
Oral
cavity cancer (squamous cell carcinoma-SCC) is one of
the most common neoplasms in the world. However, there
is no ideal animal model which can mimic many features
of human oral cavity SCC development. In the present study
we generated an animal model using the carcinogen 4-nitroquinoline
1-oxide (4-NQO) to induce tumorigenesis in the mouse oral
cavity. After 4 months, massive tumors were observed on
the tongues of CBA mice treated with 4-NQO in drinking
water. Moreover, 4-NQO treatment caused changes in retinoid
metabolism in the mouse tongues. Immunohistochemistry
analysis showed that 4-NQO changed the expression pattern
of cytokeratin14 (K14) in the epithelia of the mouse tongues.
K14 was expressed in the suprabasal layers plus the basal
layer of the tongues, unlike the controls, in which K14
was expressed only in the basal layer. 4-NQO also increased
the level of cytokeratin 1 (K1) expression. We further
observed an increase of Ki67 level after 4-NQO treatment.
These results indicate that this 4-NQO induced oral carcinogenesis
model simulates some aspects of human oral cavity carcinogenesis.
Transcriptional
activation of nuclear factor kappa B (NFkB) in ultraviolet
B (UVB)-induced basal cell carcinomas (BCCs) in ptch+/-
mice
Hong Zhang, Mohammad Athar, Xiuwei Tang, Michelle Aszterbaum,
Levy Kopelovich, Ervin H. Epstein Jr., David R. Bickers,
Department of Dermatology, College of Physicians &
Surgeons, Columbia University, New York, NY; Columbia
University, New York, NY; Department of Dermatology, College
of Physicians & Surgeons, Columbia University, New
York, NY; University of California, San Francisco, San
Francisco, CA;
Chemoprevention Division, NCI, Bethesda, MD; Department
of Dermatology, College of Physicians & Surgeons,
Columbia University, New York, NY.
BCCs
are the most common human cancer, affecting 750,000 Americans
each year and are due primarily to solar UVB exposure.
Mutational activation of the sonic hedgehog signaling
(shh) pathway is known to play an important role in BCCs
development. Ptch+/- heterozygous knockout mice develop
BCCs after chronic exposure to UVB. In these mice, we
first assessed the involvement of oxidant stress during
UVB-induced BCCs development using a novel fluorescence
method that detects reactive oxygen species (ROS) in tissue.
We observed that in control skin (non-UVB irradiated)
only hair follicles show fluorescence whereas in UVB-irradiated
non-tumor skin and in BCCs bearing skin fluorescence was
observed in epidermis and in tumor. These data confirm
increased oxidant stress during UVB-induced BCCs development.
NFkB is a ubiquitous transcription factor involved in
proliferation and tumor promotion and is known to be activated
by ROS and other stimuli known to generate ROS. The involvement
of oxidant-mediated activation of NFkB in BCCs development
was assessed by employing electrophoretic mobility shift
assay (EMSA) using the kB motif of the mouse IgGk light
chain. Increased NFkB-DNA binding occurred in both UVB-irradiated
non-tumor bearing skin and in BCCs whereas a very weak
signal was detected in non-irradiated age matched controls.
Since NFkB complexes consist of different homo- and heterodimers,
we compared NF_B complexes in control, and UVB-irradiated
non-tumor skin and BCCs bearing skin employing a gel supershift
assay. A p50 NFkB complex was seen in BCCs. UVB is known
to enhance cyclin D1 expression in human BCCs and the
promoter region of the Cyclin D1 gene contains a NFkB
binding domain. To assess whether NFkB activation is accompanied
by elevated expression of cyclin D1 in BCCs, we immunostained
control (non-irradiated), and UVB-irradiated non-tumor
skin and BCCs bearing skin and observed Cyclin D1 over-expression
in suprabasal layers of UVB-exposed non-tumor skin and
in BCCs. These results indicate that ROS and NFkB activation
accompany UVB-induced BCCs development in ptch+/- mice.
High
frequency of p16 and RASSF1A methylation and their relationship
to aflatoxin B1-DNA adducts in human hepatocellular carcinoma
Yujing Zhang, Yu Chen, Habibul Ahsan, Ruth M. Lunn, Liyu
Wang, Shuyuan Chen, Chienjen Chen, Regina M. Santella,
Columbia Univ. Cancer Center, New York, NY; School of
Public Health National Taiwan Univ., Taipei, Taiwan.
Epigenetic
changes in gene expression due to extensive CpG island
methylation is now accepted as the main cause of inactivation
of p16. More recently, it has been suggested that the
human Ras association domain family 1 (RASSF1) gene, cloned
from the lung tumor suppressor locus 3p21.3 may also be
inactivated by methylation. It consists of two major alternative
transcripts, RASSF1A and RASSF1C; epigenetic inactivation
of isoform A was observed in several carcinomas and tumor
cell lines. In this study, promoter hypermethylation of
p16 and RASSF1A was investigated in 83 hepatocellular
carcinoma (HCC) tissues from Taiwan and in two HCC cell
lines (Hep3B and HepG2). High frequencies (47% and 85%,
respectively) of methylation of the CpG island promoters
of p16 and RASSF1A were found in the HCC samples. The
methylation of RASSF1A was also detected in Hep3B cells;
p16 was not methylated in either cell line. These results
suggest that aberrant methylation of the CpG island promoters
of both genes is a frequent occurrence in hepatocarcinogenesis.
The relationship between methylation status and clinical
parameters and tumor markers including, DNA damage resulting
from three environmental carcinogens, aflatoxin B1 (AFB1),
4 aminobiphenyl and polycyclic aromatic hydrocarbons and
p53 status was also analyzed. Interestingly, a statistically
significant association was found between the RASSF1A
methylation status and the level of AFB1 DNA adducts in
tumor tissues. No association was found between methylation
status and 4 ABP or PAH DNA or p53 status. These results
suggest an interesting hypothesis that exposure to environmental
carcinogens may be involved in altered methylation of
genes involved in cancer development.
Cyclooxygenase-2:
from signal transduction to therapy
Andrew J. Dannenberg, Nasser K. Altorki, Kotha Subbaramaiah,
Weill Medical College of Cornell University and Strang
Cancer Prevention Center, New York, NY.
Cyclooxygenase (COX) catalyzes the synthesis of prostaglandins
(PGs) from arachidonic acid. Nonsteroidal anti-inflammatory
drugs (NSAIDs) inhibit COX-mediated synthesis of PGs.
There are two isoforms of COX. One is constitutive (COX-1),
and the other is inducible (COX-2). The COX-2 gene is
an immediate early response gene that is induced by growth
factors, cytokines, oncogenes and tumor promoters (1-3).
Both the Ras and protein kinase C signal transduction
pathways are important for regulating the expression of
COX-2.
There
is substantial evidence that COX-2 represents a pharmacological
target for the prevention and treatment of cancer. COX-2
is overexpressed in transformed cells and in a variety
of human malignancies (3,4). In transgenic mice, COX-2
overexpression in the mammary gland was sufficient to
cause tumor formation (5). Knocking out the COX-2 gene
led to a marked reduction in the number of intestinal
and skin tumors in experimental animals (6). In addition
to the genetic evidence implicating COX-2 in tumorigenesis,
there are supporting pharmacological data. Selective COX-2
inhibitors prevent the formation and growth of tumors
in experimental animals (6-9). Several different mechanisms
have been identified that can account for the link between
COX-2 and cancer. Enhanced synthesis of PGs, which occurs
in a variety of tumors, can increase cell proliferation,
promote angiogenesis and inhibit immune surveillance (4).
Additionally, overexpression of COX-2 in epithelial cells
inhibits apoptosis and enhances invasiveness, which are
likely to increase the tumorigenic potential of affected
cells (10,11). The relative importance of each of these
mechanisms in carcinogenesis remains to be elucidated.
Based on the strength of these preclinical findings, selective
COX-2 inhibitors are being intensively investigated as
anti-cancer
agents in humans.
1.
Kujubu, D.A., Fletcher, B.S., Varnum, B.C., Lim, R.W.,
and Herschman, H.R. TIS10, a phorbol ester tumor promoter-inducible
mRNA from Swiss 3T3 cells, encodes a novel prostaglandin
synthase/cyclooxygenase homologue. J. Biol. Chem., 266:
12866-12872, 1991.
2.
DuBois, R.N., Awad, J., Morrow, J., Roberts, L.J., and
Bishop, P.R. Regulation of eicosanoid production and mitogenesis
in rat intestinal epithelial cells by transforming growth
factor-alpha and phorbol ester. J. Clin. Invest, 93:493-498,
1994.
3.
Subbaramaiah, K., Telang, N., Ramonetti, J.T., Araki,
R., DeVito, B., Weksler, B.B., and Dannenberg, A.J. Transcription
of cyclooxygenase-2 is enhanced in transformed mammary
epithelial cells. Cancer Res., 56: 4424-4429,1996.
4.
Dannenberg, A.J., Altorki, N.K., Boyle, J.O., Dang, C.,
Howe, L.R., Weksler, B.B. and Subbaramaiah, K. Cyclo-oxygenase-2:
a pharmacological target for the prevention of cancer.
Lancet Oncol. 2:544-551, 2001.
5.
Liu, C.H., Chang, S.H., Narko, K., Trifan, O.C., Wu, M.T.,
Smith, E., Haudenschild, C., Lane, T.F. and Hla, T. Overexpression
of cyclooxygenase-2 is sufficient to induce tumorigenesis
in transgenic mice. J. Biol. Chem. 276:18563-18569, 2001.
6.
Oshima, M., Dinchuk, J.E., Kargman, S.L., Oshima, H.,
Hancock, B., Kwong, E., Trzaskos, J.M., Evans, J.F., and
Taketo, M.M. Suppression of intestinal polyposis in Apc
delta716 knockout mice by inhibition of cyclooxygenase
2 (COX-2). Cell, 87: 803-809, 1996.
7.
Kawamori, T., Rao, C.V., Seibert, K., and Reddy, B.S.
Chemopreventive activity of celecoxib, a specific cyclooxygenase-2
inhibitor, against colon carcinogenesis. Cancer Res.,
58: 409-412, 1998.
8.
Fischer, S.M., Lo, H-H., Gordon, G.B., Seibert, K., Kelloff,
G., Lubet, R.A., and Conti, C.C. Chemopreventive activity
of celecoxib, a specific cyclooxygenase-2 inhibitor, and
indomethacin against ultraviolet light-induced skin carcinogenesis.
Mol. Carcinog., 25: 231-240, 1999.
9.
Sheng, H., Shao, J., Kirkland, S.C., Isakson, P., Coffey,
R.J., Morrow, J., Beauchamp, R.D., and DuBois, R.N. Inhibition
of human colon cancer cell growth by selective inhibition
of cyclooxygenase-2. J. Clin. Invest., 99: 2254-2259,
1997.
10.
Tsujii, M., and DuBois, R.N. Alterations in cellular adhesion
and apoptosis in epithelial cells overexpressing prostaglandin
endoperoxide synthase 2. Cell, 83: 493-501, 1995.
11.Dohadwala,
M., Luo, J., Zhu, L., Lin, Y., Dougherty, G.J., Sharma,
S., Huang, M., Pold, M., Batra, R.K., and Dubinett, S.M.
Non-small cell lung cancer cyclooxygenase-2-dependent
invasion is mediated by CD44. J. Biol. Chem., 276:
20809-20812, 2001.
Tobacco
smoke induces cyclooxygenase-2 in epithelial cell lines
derived from the human aerodigestive tract
Dimitrios Moraitis, Taghreed Almahmeed, Babette Weksler,
Jay Boyle, Kazuhiko Yoshimatsu, Nasser Altorki, Fan Zhang,
Kotha Subbaramaiah, Andrew J. Dannenberg, Memorial-Sloan
Kettering Cancer Center, New York, NY; Weill Medical College,
New York, NY; Strang Cancer Prevention Center, New York,
NY.
Cyclooxygenase-2
(COX-2), the inducible form of COX, catalyzes the synthesis
of prostaglandins (PG) from arachidonic acid. Multiple
lines of evidence suggest that overexpression of COX-2
predisposes to cancer at a variety of sites. Given the
link between COX-2 and cancer, it is important to identify
exogenous as well as endogenous factors (oncogenes, cytokines)
that regulate the expression of COX-2. Individuals who
smoke tobacco are at increased risk for cancers of the
aerodigestive tract. We were interested, therefore, in
determining whether tobacco smoke (TS) induced COX-2 and
PG biosynthesis in cell lines derived from the aerodigestive
tract. TS was collected as whole mainstream cigarette
smoke bubbled through phosphate buffered saline. The addition
of dilute TS to multiple cell lines caused dose-dependent
induction of PG production without cytotoxicity. Western
and northern blotting were performed to investigate whether
the observed effects on PG synthesis reflected changes
in amounts of COX-2. Consistent with the changes in PG
production, treatment with TS caused a dose-dependent
increase in amounts of both COX-2 mRNA and COX-2 protein.
These inductive effects were observed for up to 24 hours.
Experiments were also carried out to determine whether
ERK1/2 MAPK was involved in mediating the inductive effects
of TS. Treatment with TS stimulated the activity of ERK1/2
MAPK. PD 98059, a compound that inhibits the activation
of ERK1/2 MAPK, blocked TS-mediated induction of COX-2.
To begin to define the chemical constituents of TS that
mediate these effects, we also investigated whether benzo[a]pyrene,
a polycyclic aromatic hydrocarbon present in TS, induced
COX-2. Treatment with benzo[a]pyrene led to dose-dependent
induction of COX-2. Taken together, these results indicate
that TS induces COX-2 at least, in part, by stimulating
ERK1/2 MAPK. Possibly, selective COX-2 inhibitors will
have a role in preventing TS-induced carcinogenesis.
Inactivation
of the mucin gene Muc2 causes colorectal cancer
Alessandra Fragale, Wancai Yang, Joerg Heyer, Courtney
Nicholas, Stephanie Viani, Raju Kucherlapati, Martin Lipkin,
Kan Yang, Leonard Augenlicht, Anna Velcich, Albert Einstein
College of Medicine, Montefiore Medical Center, Bronx,
NY; Strang Cancer Prevention Center, New York, NY.
Mucins,
highly glycosylated proteins, are the major component
of mucus, which lubricates and protects the underlying
intestinal epithelium. Alterations of mucin gene expression
and mucin glycosylation characterize later stages of colon
tumor
progression. However, modulation of mucin gene expression
also characterizes early stages of intestinal tumorigenesis.
To evaluate the importance of mucin in the early steps
of intestinal carcinogenesis, we genetically inactivated
the mouse Muc2gene, which encodes the most abundant secreted
gastrointestinal apomucin, a marker of goblet cells. Alcian
blue and immunohistochemical staining failed to detect
goblet cells in the intestinal tract of Muc2 knockout
mice. The absence of mucin did not completely eliminate
the goblet cell lineage differentiation since both by
in situ hybridization and immunohistochemistry we detected
Itf, a product of mature goblet cells. Further, the absence
of Muc2 did not alter the expression levels of other apomucins,
as assayed by northern blot and immunohistochemical analysis.
Muc2-/- mice displayed aberrant intestinal crypt morphology,
characterized by altered proliferation, migration and
apoptosis of epithelial cells. Muc2-/- animals developed
adenomas that progressed to invasive adenocarcinoma in
the small intestine, and rectal tumors. Tumor formation
occurred independently of alteration of the b catenin-Tcf
pathway as demonstrated by the absence of increased expression
and nuclear localization of b-catenin, abnormalities that
we detected in intestinal tumors of Apc 1638 mice. However
tumors in Muc2-/- mice were characterized by very high
expression of c-Myc and a direct target of c-Myc,
Cdk-4, suggesting that alteration of c-Myc is important
in tumor development and occurs through a mechanism distinct
from deregulation of the Apc-b catenin pathway. In summary,
Muc2 suppresses development of colorectal tumorigenesis,
suggesting that early alterations in Muc2 expression may
be involved in colorectal tumorigenesis.
Overexpression
of IL-1b gene contributes to retinoic acid-induced cell
growth inhibition of human mammary epithelial cells
Limin Liu, Lorraine Gudas, Department of Pharmacology,
Weill Medical College of Cornell University, New York,
NY.
We
identified interleukin-1 b (IL-1 b) gene as a retinoic
acid (RA) up-regulated gene in normal mammary epithelial
cells; its effects on the growth of normal human mammary
epithelial cells (HMEC) and breast carcinoma cell lines
were examined. To identify the genes that mediate RA-induced
cell growth arrest, a cDNA library was synthesized from
HMEC polyA+ mRNA and subtractive screening was performed.
The IL-1 b gene was cloned as a RA regulated gene. Northern
blot analyses indicated that the IL-1 b gene was up-regulated
as early as 2 hours after RA treatment. Data from the
treatment of HMEC with cycloheximide and actinomycin D
indicated that the IL-1 b gene was a direct downstream
target gene of RA receptors and that the regulation of
IL-1 b occurred at the transcriptional level. The HMEC
expressed both type 1 and type 2 IL-1 receptors. To evaluate
the effect of IL-1 b on cell growth, the growth of HMEC
was observed in the presence of RA (1uM) or IL-1 b (100pg/ml),
or both. The results showed that either RA or IL-1 b could
significantly inhibit the growth of HMEC. The growth inhibition
was even more marked when HMEC were treated with RA +
IL-1 b. The expression of IL-1 b was examined in the breast
carcinoma cell lines MCF-7, T47D, SKBR-3, MB-231 and MB-468
with and without RA treatment, and the Northern blot analysis
indicated that IL-1 b expression was largely abolished
in all of the carcinoma cell lines as compared to the
HMEC. Growth curves of the carcinoma cell lines showed
that the estrogen receptor (ER) positive MCF-7 cell line
responded to RA and IL-1 b treatment, while neither RA
nor IL-1 b inhibited the growth of the ER negative MB-231
cells. Studies on other tissues indicated that the expression
of the IL-1 b gene was also up-regulated by RA in the
epithelial cells of prostate and oral cavity. Our results
suggest: 1. The IL-1 b gene is a direct target of RA receptors
and its overexpression may contribute to RA-induced HMEC
growth arrest. 2. The expression of the IL-1 b gene is
low or absent in all examined breast carcinoma cells,
but the malfunction of IL-1 b signaling pathway may occur
at different levels in ER positive vs. ER negative carcinoma
cells.
The
roles of Stat3 in cyclin D1 expression, cell proliferation
and chemosensitivity in head and neck squamous cell carcinoma
cells
Muneyuki Masuda, Masumi Suzui, Jin T. E. Lim, Sohtaro
Komiyama, I. Bernard Weinstein, Herbert Irving Cancer
Center, Columbia University, New York, NY; Department
of ORL, Faculty of Medicine, Kyushu University, Fukuoka,
Japan.
Overexpression
of the important cell cycle control protein cyclin D1,
and or the related mRNA, occurs in over 50% of cases of
human head and neck squamous cell carcinoma (HNSCC), and
overexpression of this protein is a marker of poor prognosis
in this disease. In about 20% of the cases of HNSCC the
cyclin D1 gene is amplified. However, in the majority
of these cases the gene is not amplified, suggesting that
the increased expression of cyclin D1 is due to defects
at the level of gene transcription. In view of the fact
that signal transducer and activator of transcription
3 (Stat3) is activated by autocrine activation of TGF-a/EGFR,
which is often found in HNSCC, we examined the effects
of a dominant negative mutant of Stat3 on cyclin D1 expression
and cell proliferation in the YCU-H891 HNSCC cell line,
since it displays constitutive activation of Stat3. In
transient transfection assays with a cyclin D1 luciferase-reporter
construct, cotransfection of the dominant negative Stat3
construct, but not treatment with a MEK-1 inhibitor, significantly
inhibited cyclinD1 promoter activity. In derivatives of
YUC-H891 cells that stably express the dominant negative
Stat3 protein, cyclin D1-luciferase promoter activity,
and cellular levels of cyclin D1 mRNA and cyclin D1 protein
were strongly inhibited when compared to parental or vector
control cells. In addition, these derivatives displayed
slower growth rates, lower saturation densities, decreased
levels of the antiapoptotic proteins Bcl-2 and B cl-XL,
and a marked (55 to 75 fold) enhancement in sensitivity
to growth inhibition by 5-fluorouracil. This study provides
the first evidence that overexpression of cyclin D1 in
HNSCC is due, at least in part, to activation of Stat3.
This result, taken together with our other findings related
to Stat3, suggest that agents that inhibit Stat3 may be
useful in the therapy of HNSCC.
Role
of Cyp26 in stem cell differentiation
Simne Ng, Michelle Lane, Lorraine Gudas, Dept of Pharmacology,
Weill Graduate School of Cornell University, New York,
NY; Weill Graduate School of Cornell University, New York,
NY.
Retinoids
and their metabolic derivatives function as regulators
of cellular differentiation, proliferation, and apoptosis.
The levels of retinoids and their metabolites are kept
in balance in the cell by their biosynthesis and catabolism.
Cyp26, a cytochrome P450 family member, is a retinoic
acid hydroxylase responsible for the metabolism of retinol
(Rol) and retinoic acid (RA) into their more polar, bioactive
metabolites. Cyp26 is activated in some types of cancer
cells, such as MCF-7 cells, by RA (Chen et al, Cancer
Res. 1997). Our previous research established that the
removal of Leukemia Inhibitory Factor (LIF), a cytokine
that blocks differentiation of murine embryonic stem (ES)
cells, induced expression of Cyp26 by 15-fold and increased
the metabolism of Rol and RA into their more polar derivatives.
To further investigate the role of Cyp26 in the differentiation
of ES cells, we are generating ES cell lines that lack
or overproduce Cyp26 in a tetracycline-inducible system.
To determine the mechanism by which Cyp26 expression is
regulated, we are constructing a luciferase reporter driven
by the Cyp26 promoter. This construct will be used to
determine how the Cyp26 gene is transcriptionally regulated
upon the removal of LIF.
Localization
of protein kinase C isoforms in living cells using green
fluorescent protein as a marker
Jae-Won Soh, I. Bernard Weinstein, Columbia University
Cancer Center, New York, NY.
Protein
kinase C (PKC) is a multigene family of enzymes consisting
of at least 11 isoforms. Specific isoforms of PKC play
key roles in pathways of signal transduction, growth control
and tumorigenesis. Variations in the intracellular localization
of
individual isoforms are thought to be an important mechanism
for isoform specific regulation of enzyme activity and
also substrate specificity. To provide a dynamic method
for analyzing the localization of specific isoforms of
PKC in living cells,
we generated fluorescent fusion proteins of PKCs-alpha,
beta1, beta2, gamma, delta, epsilon, eta, zeta and iota,
using the green fluorescent protein (GFP) as a fluorescent
marker at the carboxyl termini of these enzymes. Intracellular
localization of specific isoforms of PKCs was then examined
by fluorescence microscopy after transient transfection
of the respective PKC-GFP expression vector into NIH3T3
mouse fibroblasts. The intracellular translocation of
specific PKC isoforms induced by TPA was also monitored
by fluorescence microscopy in real time. We found that
specific isoforms of PKC display distinct localization
patterns in untreated NIH3T3 cells. For example, PKC-alpha
is localized mainly in the cytoplasm and PKC-epsilon is
localized mainly in the Golgi apparatus. We also observed
that PKC-alpha, beta1, beta2, gamma, delta, epsilon and
eta translocate to the plasma membrane within 10 minutes
of TPA treatment, while the cellular localization of PKC-zeta
and iota are not affected by TPA. These results suggest
that these isoform-specific PKC-GFP fusion proteins may
be useful markers for determining the localization of
specific isoforms of PKCs in various types of living cells,
and for elucidating dynamic changes in response to various
stimuli, or cell transformation.
Augmented
basal cell carcinomas development in ptch+/- heterozygous
knockout mice over-expressing ornithine decarboxylase
Mohammad Athar, Justin Russo, Michelle Aszterbaum, Arianna
L. Kim, Hong Zhang, Xiuwei Tang, Levy Kopelovich, Ervin
H. Epstein Jr., David R. Bickers, Department of Dermatology,
College of Physicians & Surgeons, Columbia University,
New York, NY; Department of Dermatology, University of
San Francisco, San Francisco, CA; National Cancer Institute,
Division of Cancer Chemoprevention, Bethesda, MO.
BCCs
of the skin are the most common human cancer. In-depth
understanding of the molecular mechanisms of BCCs induction
has been hampered by the lack of an appropriate animal
model. Based upon discoveries in patients with the dominantly
inherited nevoid basal cell carcinoma syndrome (NBCCS),
mutations in sonic hedgehog (shh) signaling genes including
patched (ptch) are known to underlie BCCs development.
ptch+/- knockout mice develop BCCs and trichoblastomas
after chronic exposure to solar ultraviolet B (UVB) radiation.
UVB is a potent inducer of cutaneous ornithine decarboxylase
(ODC) activity, which drives the continued proliferation
and clonal expansion of initiated cells leading to tumor
development, we reasoned that over-expressing ODC in ptch+/-
heterozygous mice might provide a model for enhancing
BCCs development. We have generated ptch+/- mice over-expressing
the ODC transgene, hereafter referred to as ptch+/-/ODC
TgN. Untreated skin at the age of seven months appeared
grossly normal; however, histologic examination shows
small basaloid lesions. Immunohistochemical studies show
positive staining for b-galactosidase (b-gal), gli-1 and
shh. None of the wild-type or ptch+/- littermates manifest
such lesions or positive staining for b-gal. Our parent
ptch+/- are heterozygous for deletion of exon 1 and 2
and insertion of lacZ and neo genes. BCCs in ptch+/-/ODC
TgN mice lead to de-repressed transcription of the lacZ
gene inserted in the inactivated ptch locus. Therefore,
positive b-gal staining demonstrates activation of shh
signaling in skin/BCCs -like lesions in ptch+/-/ODC TgN
mice. Chronic UVB irradiation (180mJ/cm2 twice a week)
for ten weeks resulted in multiple small visible tumors,
which increased both in number and size after another
twenty weeks of irradiation. Tumor incidence was 100%
by week 20. Histologically, these lesions resemble human
BCCs and trichoblastomas and show strong positive staining
for b-gal, gli-1 and shh. Our data show that b-gal positive
staining lesions also stain for gli-1/shh. These results
indicate that ptch+/-/ODC TgN mice are a sensitive model
for studying the pathogenesis of UVB-induced BCCs.
The
role of protein kinase G (PKG) in proliferation, migration
and apoptosis in human colon cancer cells
Atsuko Deguchi, Jae-Won Soh, Han Li, Rifat Pamukcu, W.
Joseph Thompson, I. Bernard Weinstein, Herbert Irving
Comprehensive Cancer Center, Columbia University, New
York, NY; Cell Pathways, Inc., Horsham, PA.
Recent
studies provide evidence that exisulind and two potent
derivatives, CP461 and CP248, induce apoptosis in colon
cancer cells by activating PKG through a mechanism involving
cGMP-phosphodiesterase inhibition. PKG Ia and Ib are ubiquitously
expressed in mammalian cells. To directly examine their
effects on growth and apoptosis we constructed a series
of mutants of PKG Ia: PKG Ia S65D, a constitutively activated
point mutant; PKG IaD, a constitutively activated N-terminal
truncated mutant; and PKG Ia K390R, a dominant negative
(DN) point mutant. A similar series of mutants of PKG
Ib were also constructed, i.e., PKG Ib S80D, PKG IbD and
PKG IbK405R. When transiently transfected into SW480 cells,
the constitutively activated mutants of PKG caused b-catenin
phosphorylation and a decrease in the level of b-catenin,
inhibition of cell migration in matrigel assays, and increased
apoptosis detected by DNA flow cytometry. We were not
able to obtain derivatives of SW480 cells that stably
expressed the constitutively activated mutants of PKG,
presumably because of toxicity. However, derivatives that
stably overexpressed wild-type PKGs Ia or Ib displayed
growth inhibition and increased sensitivities to induction
of apoptosis by a cell-permeable form of cGMP. In contrast,
derivatives that stably expressed dominant negative PKG
Ia or Ib grew more rapidly and were more resistant to
exisulind-induced apoptosis. Taken together, these results
provide direct evidence that activation of PKG Ia or Ib
in human colon cancer cells can inhibit both cell proliferation
and cell migration and enhance the induction of apoptosis.
Therefore, PKGs Ia or Ib provide novel molecular targets
for cancer chemoprevention and therapy.
G2/M
cell cycle arrest by the pro-apoptotic cGMP phosphodiesterase
inhibitor, CP248, in SW480 colon cancer cells involves
inhibition of tubulin polymerization via the colchicine
binding site
John R. Fetter, Clark Whitehead, Jung-Taek Yoon, Danhua
Xiao, Gregg G. Gundersen, I. Bernard Weinstein, Gerhard
Sperl, Rifat Pamukcu, W. Joseph Thompson, Cell Pathways,
Horsham, PA; Herbert Irving Comprehensive Cancer Center,
New York, NY.
Previous studies have shown that exisulind and a high
affinity analog CP248 inhibit cGMP phosphodiesterases
and activate protein kinase G, leading to decreases in
b-catenin, activation of JNK1, and induction of apoptosis
(W. J. Thompson, et al.,
Cancer Res. 60, 3338-3342, 2000; J. W. Soh et al., Clin.
Cancer Res. 6, 4136-4141, 2000). SW480 colon cancer cells
treated with 0.2 mM CP248 for 24 hours showed a 70% accumulation
in G2/M relative to 18% for the control. Analysis with
the MPM2 antibody indicated that 22% of the cells were
in the M phase compared to 3% for the control. Immunofluorescent
staining for microtubules using a monoclonal antibody
to a-tubulin revealed complete dissolution of the microtubule
cytoskeleton. CP248 contains a trimethoxy benzyl group
similar to that found in the tubulin-binding compounds
colchicine and colcemid, agents that inhibit tubulin polymerization
and cause G2/M arrest. CP248 was tested for a direct effect
on tubulin polymerization as measured by an in vitro turbidity
assay and was found to inhibit the rate of tubulin polymerization
when using either pure tubulin or MAP-rich tubulin. To
further characterize the tubulin interaction, CP248 was
tested for competition with fluorescent colchicine. CP248
at 10 mM was shown to inhibit 50% of fluorescent colchicine
binding to tubulin, similar to the effect seen with 10
mM colchicine. The Ki for colchicine binding is 0.3-2.0
mM, similar to the concentrations at which CP248 induces
apoptosis and inhibits cGMP phosphodiesterases, 0.6 mM
and 0.3 mM respectively. We conclude that high affinity
analogs of exisulind like CP248 may offer advantages for
growth inhibition and apoptosis
induction in neoplasia since direct inhibition of tubulin
polymerization is combined with cGMP-mediated pathways
of apoptosis.
p27
and cyclins are involved in RARb-mediated growth arrest
by retinoic acid in murine F9 teratocarcinoma cells
Rong Li, Teresa N. Faria, Pierre Chambon, Lorraine J.
Gudas, Weill Medical College of Cornell University, New
York, NY; Institut de Genetique et de Biologie Moleculaire
et Cellulaire, Illkirch Cedex, France.
It
has been suggested that RARb plays an important role in
mediating the anticarcinogenic effects of retinoic acid
(RA). We have previously shown that RARb2 knockout murine
F9 teratocarcinoma cell lines have completely lost responsiveness
to
RA-induced growth arrest. In this study we examined the
cell cycle regulators affected by RA in F9 wild type (Wt)
and RARb2-/- F9 cells and the mechanisms by which RARb2-/-
F9 cells become refractory to the growth inhibitory action
of RA. Murine F9 Wt cells and the RARb2-/- F9 line were
cultured in the presence or absence of all-trans retinoic
acid (1 mM, Sigma, St Louis, MO). The cell cycle distribution
was analyzed by flow cytometry. After RA treatment for
96 hours the F9 Wt cells shifted their cell cycle distribution
profile by increasing the percentage of cells in G1. In
contrast, the RARb2-/- F9 cell lines did not change their
cell cycle distribution profile after RA treatment. The
expression of mRNAs encoding cyclin D1, CDK2 and p27 proteins
was determined in F9 Wt and RARb2-/- cells after RA treatment
for up to 96 hours. The levels of cyclin D1 mRNA decreased
in both cell lines after RA treatment, while there was
no change in the levels of CDK2 and p27 mRNAs in either
F9 Wt or RARb2-/- cells after treatment with RA. We then
assayed the protein levels of G1 cell cycle regulators
in F9 Wt and RARb2-/- cells after RA treatment for up
to 96 hours. In F9 Wt cells, cyclin D1, D3 and cyclin
E levels decreased with RA treatment, while cyclin D2
and p27 increased after RA treatment. Unlike the F9 Wt
cells, RA treatment did not change the levels of the above
cyclins and p27 in the RARb2-/- F9 cells. Furthermore,
we found that the level of p27 was regulated at the post-translational
level. The level of other cell cycle regulators did not
change appreciably with or without RA treatment in either
of the two cell lines. These included CDK2, CDK4, CDK6
and p21. Considering the possible roles that RARb plays
in the process of carcinogenesis and in the mediation
of RA-induced growth arrest in a variety of cancer cells,
our findings delineate some of the molecular mechanisms
by which RARb mediates the growth inhibitory effects of
RA.
A
SWI/SNF family member gene is frequently silenced in human
colon cancer
Helen R. Moinova, Weidong Chen, Lanlan Shen, Dominic Smiraglia,
Joseph Olechnowicz, Lakshmeswari Ravi, Lakshmi Kasturi,
Lois Myeroff, Christoph Plass, Ramon Parsons, John Minna,
James K. V. Willson, Sylvan B. Green, Jean-Pierre Issa,
Sanford D. Markowitz, Ireland Cancer Center and Case Western
Reserve University,
Cleveland, OH; Department of Leukemia, University of Texas
at MD Anderson Cancer Center, Houston, TX; Division of
Human Cancer Genetics, Ohio State University, Columbus,
OH; Howard Hughes Medical Institute, Cleveland, OH; Department
of Pathology, Columbia University, New York, NY; The Hamon
Center for Therapeutic Oncology Research, University of
Texas Southwestern Medical Center at Dallas, Dallas, TX;
Cancer Center and Department of Medicine at Case Western
Reserve University, and Ireland Cancer Center, Department
of Medicine, and Research Institute at University Hospitals
of Cleveland, Cleveland, OH; Cancer Center and Department
of Epidemiology and Biostatistics, Case Western Reserve
University, Cleveland, OH;
Howard Hughes Medical Institute and Case Western Reserve
University Ireland Cancer Center, Cleveland OH.
Chromatin
remodeling enzymes play an important role in a variety
of cellular functions. Mutations of several components
of chromatin remodeling complexes, including members of
SWI/SNF family, have been identified in cancer. In this
study we describe SWI/SNF/MCC (SWI/SNF methylated in colon
cancer). SWI/SNF/MCC is a helicase-like transcription
factor that is a member of the SWI/SNF gene superfamily
and that is a novel target for gene inactivation in colon
cancer. Loss of the expression was noted in 9 out of 34
colon cancer cell lines and in 27 of 63 cases (43%) of
primary colon cancer tissues. Methylation of CpG island
in the SWI/SNF/MCC promoter correlated with loss of gene
expression and SWI/SNF/MCC mRNA was re-expressed upon
treatment with demethylating agent 5-azacytidine. No methylation
of SWI/SNF/MCC was detected in breast or lung cancers,
suggesting selection for SWI/SNF/MCC methylation in colonic
malignancies. Transfection of SWI/SNF/MCC into an SWI/SNF/MCC
deficient cell line suppressed 77% of colony growth, but
showed no suppressive effect in an SWI/SNF/MCC proficient
cell line. These findings show that SWI/SNF/MCC is a common
target for methylation and epigenetic gene silencing in
colon cancer, and suggest HLTF is a candidate colon cancer
suppressor gene.
Growth
inhibition of human hepatoma cells by acyclic retinoid
is associated with inhibition of expression of cyclin
D1
Masumi Suzui, Muneyuki Masuda, Jin T. E. Lim, I. Bernard
Weinstein, Herbert Irving Comprehensive Cancer Center,
College of Physicians and Surgeons, Columbia University,
New York, NY.
Acyclic
retinoid (ACR), a novel synthetic retinoid, can prevent
the recurrence of hepatomas after surgical resection of
primary tumors, but the molecular mechanisms by which
ACR exerts anti-tumor effects are not known. In this study
we found that ACR inhibited the growth of human hepatoma
cell lines. This was associated with an arrest of the
cell cycle in G0/G1, increased levels of p21WAF1/CIP1,
decreased levels of hyperphosphorylated Rb, and decreased
levels of cyclin D1, but no significant changes in the
levels of the p16INK4a, p27KIP1, cdk4, cdk6, GSK-3b, and
b-catenin proteins. ACR also caused a decrease in the
level of cyclin D1 mRNA. Co-treatment of HepG2 human hepatoma
cells with the proteasome inhibitor LLnL did not prevent
the ACR-induced decrease in the cyclin D1 protein, in
contrast to the protective effect of LLnL on the cyclin
D1 protein seen in cells treated with the all-trans retinoic
acid (ATRA). Furthermore in transient transfection reporter
assays, we obtained evidence that ACR, but not ATRA, inhibits
transcription from the cyclin D1 promoter. Taken together,
these findings suggest that ACR causes a decrease in the
levels of expression of the cyclin D1
protein not through proteolysis but by causing a decrease
in the level of cyclin D1 mRNA. We found that in hepatoma
cells, as previously described in colon carcinoma cells,
cyclin D1 promoter activity is stimulated by the b-catenin/TCF
pathway.
ACR also markedly inhibited this stimulation but also
exerted other inhibitory effects on cyclin D1 transcription.
These novel effects of ACR suggest that this agent might
be useful in the chemoprevention and therapy of various
malignancies.
Antiproliferative
effects of garlic-derived S-allylmercaptocysteine (SAMC)
are associated with microtubule depolymerization
Danhua Xiao, Jung T. Yoon, Alexander Palazzo, Gregg G.
Gundersen, John T. Pinto, Haim Shirin, I. Bernard Weinstein,
Irving Comprehensive Cancer Center, Columbia University,
New York, NY; Dept. of Anatomy and Cell Biology, Columbia
University, New York, NY; American Health Foundation,
Valhalla, NY; The E. Wolfson Medical Center, Holon, Israel.
Epidemiological
and experimental carcinogenesis studies provide evidence
that components of garlic (Allium sativum) have anti-cancer
activity. We recently reported that the water-soluble
garlic derivative SAMC, but not S-allylcysteine (SAC),
inhibits growth, arrests cells in G2/M, and induces apoptosis
in SW480 and HT29 human colon cancer cells (Shirin et
al, Cancer Res. 61, 725-731, 2001). Since a fraction of
the SAMC treated cells are specifically arrested in mitosis,
in the present study we examined the mechanism of this
effect. Immunostaining with the rat-YL1/2 (tyrosinated
a-tubulin) antibody and a goat anti-rat IgG-FITC conjugate
revealed that treatment of SW480 cells or NIH3T3 fibroblasts
with 150 mM SAMC (the IC50 concentration) caused microtubule
depolymerization. In vitro turbidity assays indicated
that SAMC acts directly on tubulin to cause microtubule
depolymerization and that SAMC also inhibits the initiation
of de novo tubulin polymerization. To investigate further
in SW480 cells the effects of allylsulfides and cysteinyl
derivatives on cell cycle parameters and microtubule assembly,
studies were conducted using two lipid-soluble allium
derivatives, diallylsulfide (DAS) and diallyldisulfide
(DADS), and several cysteinyl analogues, namely, cysteine,
cystine, reduced and oxidized glutathione, S-propyl glutathione,
and S-trityl-L-cysteine. Of these compounds, only DADS
and trityl-cysteine inhibited cell growth, with IC50 values
of 56 and 0.9 mM, respectively. Both compounds induced
apoptosis and arrested cells in mitosis. By contrast to
effects with SAMC, microtubule depolymerization was not
observed. These studies suggest that an active allyl and
a disulfide moiety may be responsible for growth inhibition
and induction of apoptosis exhibited by SAMC and related
compounds. SAMC may exert anti-proliferative effects,
at least in part, by disrupting microtubule assembly thus
arresting cells in metaphase and triggering downstream
signaling pathways that lead to apoptosis.
Inhibition
of prostaglandins by celecoxib results in suppression
of tumor growth and reduces VEGF levels in human head
and neck xenograft model
Ben Scott Zweifel, Richard Ornberg, Mark Woerner, Alane
Koki, Jaime Masferrer, Andrew J. Dannenberg, Jay Boyle,
Pharmacia Corp., St. Louis, MO; Cornell University, New
York, NY; Memorial Sloan-Kettering, New York, NY.
The
anti-tumor efficacy of celecoxib was demonstrated in a
novel xenograft model that expresses COX-2 in both stromal
(inflammatory and neovascular) and neoplastic cells, similar
to the pattern observed in human cancer. Human head and
neck 1483 cells were implanted in the hind paw of athymic
nude mice and tumors grew to 1.0 – 1.2 ml within 4 weeks.
Compared to vehicle treated controls, celecoxib treatment
(40-160 ppm) resulted in a dose-dependent reduction in
tumor prostaglandin E2 levels (124.9+42.3 vs 11.7+2.1
ng/gm at 160ppm) that correlated with a similar reduction
in tumor growth (1.1+0.1 vs 0.3+0.03 ml). This model is
PGE2-dependent since an inactivating PGE2 antibody showed
equivalent tumor control as celecoxib. In contrast to
the specific COX-2 inhibitor celecoxib, treatment with
SC-560, a selective COX-1 inhibitor, had no effect on
either tumor prostaglandin levels or growth. The inhibition
of tumor growth by celecoxib resulted in a 50% reduction
in cell proliferation (BrdU labeling index) and a 2 fold
increase in the apoptosis (TUNEL labeling index) observed
both in tumor and endothelial/stromal cells after 12 days
of treatment. A significant reduction in VEGF staining
and levels was observed on 2 days and 12 days (40% and
>80% of control tumor levels, respectively) in celecoxib-treated
tumors. In addition, untreated tumor-bearing animals developed
hypercalcemia (9.9 vs 14.2 mg/dl) and lost body weight
(18%) compared to normal non-tumor bearing animals. Celecoxib
treatment prevented hypercalcemia (9.4+ 0.4 mg/dl) and
weight loss throughout the study (22.3+0.3 gm vs 29.5+1.4
gm). In conclusion, the head and neck 1483 xenograft model
mimics
COX-2 expression and presents symptoms found in human
cancer. Celecoxib treatment of 1483 xenograft-bearing
mice dose-dependently reduced PGE2 levels, resulting in
tumor control and reduced tumor VEGF levels, serum calcium
levels and maintenance of body weight.
Identification
and characterization of RAR beta target genesA
Yong Zhuang, Teresa Faria, Lorraine Gudas, Weill Medical
College of Cornell University, New York, NY.
Retinoids
inhibit cell growth and promote cell differentiation.
Some retinoids have been used pharmacologically for cancer
prevention and cancer treatment. It has been widely observed
that expression of RARb, which is one of the six retinoid
receptors, is lost or reduced in many cancers. Much data
suggests that the loss or reduction of RARb expression
is an important event in tumorigenesis. To date, no genes
that are exclusively targets of RARb have been identified.
The identification of such genes should provide information
concerning the mechanism by which RARb expression inhibits
tumor development. Thus, we carried out experiments to
identify RARb targets. F9 wild type teratocarcinoma cells
and RARb-/- cells, generated by homologous recombination,
were treated with RA for 24 hours and cDNA was prepared
from these cells. Subtractive hybridization and gene microarray
screens were used to identify RARb target genes in F9
cells. Many putative RARb targets were identified, including
transcription factors, protein tyrosine kinases, homeobox
proteins, oncoproteins, ion channels, etc. Northern blot
analyses were used to examine the regulation of some of
these target genes by RA in F9 Wt cells. The regulation
of these genes was also examined by Northern blot analysis
in the F9 RARb-/-, RARb-/-, RARb-/- cell lines. Additionally,
we also examined the expression of some of these RARb
target genes in several cancer cell lines and several
tissues from Wt and RARb-/- mice.
Chemoprevention
of basal cell carcinomas in the ptc1 +/- mouse
Michelle Aszterbaum, Jennifer Hebert, Ken Lee, Mohammed
Athar, Levy Kopelovich, David Bickers, Ervin Epstein Jr.,
University of California San Francisco, San Francisco,
CA; Columbia University, New York, NY; National Cancer
Institute, Bethesda, MD.
Basal cell carcinomas are the commonest human cancer,
and their incidence is rising inexorably. In the past,
their study has been hampered by the limitation of production
of skin cancers to the squamous but not basal cell lineage
following carcinogenic insults to mouse skin. We now report
results of our initial assessment of the efficacy of three
classes of chemopreventive agents - tea, NSAIDs, and retinoids
- vs BCC development in the skin of ptc1 +/- mice, which
mimic PTCH1 +/- human (basal cell nevus syndrome) patients
in their susceptibility to basal cell carcinogenesis.
(1) We find that feeding of black or green tea or of a
partially-purified extract of green tea (polyphenone E)
has little effect on UV-induced BCCgenesis. High dose
feeding of EGCG, the putatively most active chemopreventive/antioxidant
moiety in green tea, does reduce tumorigenesis in this
model. (2) Unlike the efficacy of NSAIDs in blocking SCC
formation in murine skin treated with UV (both as reported
by others and as found by us in hairless mice), neither
a non-specific cox 1/2 inhibitor (sulindac) nor a selective
cox 2 inhibitor (celecoxib) block BCC formation induced
by UV or by ionizing radiation. In fact, contrary to our
expectations, celecoxib fed mice appear to have a trend
towards enhanced BCCgenesis. (3) By contrast, topical
application of the receptor-specific retinoid tazarotene
during the course of UV radiation blocks BCC formation
by approximately 90%, and oral administration of this
retinoid blocks the growth of microscopic tumors induced
by previous UV radiation. Thus this model appears to offer
a novel and unique resource for the development of chemopreventive
agents that will block BCC formation in BCNS patients
and, hopefully, in the general population.
Blockade
of hedgehog signaling inhibits ultraviolet B (UVB)-induced
basal cell carcinomas (BCCs) in ptch+/- knockout heterozygous
mice
Arianna L. Kim, Mohammad Athar, Xiuwei Tang, Michelle
Aszterbaum, Levy Kopelovich, Ervin H. Epstein Jr., David
R. Bickers, Columbia University, New York, NY; University
of California, San Francisco, CA; National Cancer Institute,
Bethesda, MD.
BCCs
induced by solar UVB are the most common form of human
cancer, affecting 750,000 Americans each year. Mutations
in sonic hedgehog (shh) signaling genes including patched
(ptch), shh and smoothened (smo) activate transcription
factors of the Gli family that regulate target genes ultimately
leading to BCCs development. Ptch+/- knockout mice develop
BCCs and trichoblastomas following chronic exposure to
UVB radiation and have an activated shh signaling pathway.
In various animal models, shh signaling is critical for
morphogenesis and cyclopamine, a specific inhibitor of
shh, alters morphogenesis. In this study we evaluated
the effect of orally administered cyclopamine on UVB-induced
BCCs development in ptch+/- mice. For this experiment,
mice were irradiated with UVB (240mJ/cm2 three times a
week) for thirty five weeks at which time 50% of the mice
had one or more BCCs. At this time point, UVB irradiation
was stopped and the mice were divided into two groups
of thirty animals each with approximately equal numbers
of tumors. Group-1 mice received no treatment whereas
group-2 mice received 0.1mg % cyclopamine (as cyclodextran
complex) in drinking water and the number of tumors was
recorded weekly. Mice treated with cyclopamine developed
35% fewer tumors compared to untreated controls by week
45. In these mice we also assessed the effect of cyclopamine
treatment on the induction of cell cycle regulatory proteins
as surrogate biomarkers of carcinogenic insult. Using
western blot analysis, high expression of cyclins D1,
A2 and B1 was observed in UVB-irradiated non-tumor bearing
skin of these mice; however in the cyclopamine treated
group a significant reduction in the expression of these
proteins occurred. These results indicate that inhibitors
of shh signaling block UVB-induced BCCs development in
ptch+/- heterozygous knockout mice.
A
western-style diet induces atypical hyperplasias in mammary
gland of normal C57Bl/6 mice
Naoto Kurihara, Yanhui Liu, Kunhua Fan, Hiroharu Shinozaki,
Kan Yang, Harold Newmark, Martin Lipkin, Strang Cancer
Prevention Center and Weill Medical College of Cornell
University, New York, NY.
Decreased
dietary calcium and vitamin D and increased fat intake
have been associated with increased risk of mammary cancer
in humans. We previously reported that a Western-style
style diet that mimics these nutrient alterations induced
hyperproliferation and hyperplasia of mammary ductal epithelium
in normal C57Bl/6 mice. We have now studied the effects
of a new Western-style diet(NWD)which also contained additional
risk factors including decreased folic acid and other
nutrients required for methyl group biochemical transfer
systems. After 3 months of feeding the NWD, 8 of 10 mice
(80%) on NWD developed atypical hyperplasias and none
were seen in 10 control mice (P<0.001). Cyclin D1 expression
also was observed in atypical hyperplastic mammary duct
epithelial cells. Morphometric studies demonstrated a
significantly increased
number of terminal ductules in mammary gland after NWD
feeding (p<0.001). Significant increases (p<0.001)
also were seen in the numbers of epithelial cells, apoptotic
cells and mitotic figures in large, intermediate, or terminal
mammary ducts of mice fed NWD compared to controls, and
cell proliferation increased 5.4-fold in BrdU labeled
cells in terminal ductules. These findings demonstrate
significant Western-style diet induction of atypical hyperplastic
lesions in mammary gland of normal C57Bl/6 mice.
a-difluoromethyl
ornithine(DFMO), an irreversible inhibitor of ornithine
decarboxylase, abrogates ultraviolet B (UVB)-induced skin
carcinogenesis in SKH-1 hairless mice
Justin E. Russo, Mohammad Athar, Xiuwei Tang, Michelle
Aszterbaum, Levy Kopelovich, Ervin H. Epstein Jr., David
R. Bickers, Department of Dermatology, College of Physicians
and Surgeons, Columbia University, New York, NY; Department
of Dermatology, University of California, San Francisco,
CA; National Cancer Institute, Division of Cancer Prevention,
Bethesda, MD.
UVB
is a known risk factor for nonmelanoma skin cancers (NMSC)
in humans. Extensive experimental evidence indicates that
cutaneous UVB exposure induces the activity of ornithine
decarboxylase (ODC), which drives the continued proliferation
and clonal expansion of initiated cells thereby playing
a crucial role in tumorigenesis. DFMO is an ornithine
analog that irreversibly inhibits ODC activity. DFMO is
effective in reducing skin tumors in various murine models
of carcinogenesis. In this study, we assessed the anti-carcinogenic
effect of orally administered DFMO against UVB-induced
skin carcinogenesis in SKH-1 hairless mice. DFMO was administered
in drinking water during chronic UVB exposure of mice.
For this experiment, sixty SKH-1 hairless mice were equally
divided into two groups. Group-I received no agent and
group-II received 1% DMFO in drinking water. Beginning
fifteen days after treatment with DFMO, these mice were
exposed to UVB (180 mJ/ cm2, twice weekly) while continuing
oral DFMO for forty weeks. By week 18, 66% of Group-I
developed tumors whereas only 3% of the Group-II animals
developed tumors. Similarly, the number of tumors/mouse
was 1.03 in UVB-treated controls (Group-I) whereas it
was 0.01 in the DFMO-treated mice (GroupII). At weeks
28 and 40, the percent tumor incidence was 97% and 100%
respectively in the UVB-treated controls (Group-I) whereas
it was 23% and 77% respectively in the DFMO-treated mice
(Group-II). The number of tumors/mouse was 3.6 and 7.1
respectively in Group-I whereas it was 0.43 and 3.1/mouse
respectively in Group-II. For studying the effect of DFMO
on malignant progression of UVB-induced tumors, mice were
irradiated with 180 mJ/cm2 UVB (two times/week) for 28
weeks. At 28 weeks, sixty animals with 5 or more tumors
were withdrawn (no further exposure to UVB) and divided
into two groups. Group-1 mice (control) received water
whereas group-2 mice received DFMO (1%). Mice exposed
to UVB for 28 weeks mainly developed SCCs when assessed
after 43 weeks. Mice receiving DFMO showed no increase
in tumor number and a 168% increase in tumor volume above
baseline compared to controls, which showed 180% increase
in tumor number and 325% increase in tumor volume. These
studies indicate that DFMO is a potent anticancer agent
in UVB-treated SKH-1 mice.
Comparative
effects of COX-1 and COX-2 inhibitors on UVB photocarcinogenesis
in SKH-1 mice
Xiuwei Tang, Mohammad Athar, Mitchelle Aszterbaum, Levy
Kopelovich, Ervin H. Epstein Jr., David R. Bickers, Department
Dermatology, College of Physicians & Surgeons, New
York, NY; Department of Dermatology, University of San
Francisco, San Francisco, CA; Division of Chemoprevention,
National Institute of Cancer, Bethesda, MD.
Ingestion
of non-steriodal anti-inflammatory drugs (NSAIDS) is known
to diminish malignant transformation of colonic polyps
in human subjects at increased risk for cancer. Inhibition
of the enzyme cyclooxygenase (COX), particularly COX-2,
is also thought to play a role in this anti-carcinogenic
effect in various epithelial cancers including skin. We
previously showed that COX-2 is overexpressed in UVB-inducedsquamous
cell carcinomas (SCCs). In addition, celecoxib, a specific
inhibitor
of COX-2 has been shown to inhibit UVB-induced papillomas
in murine skin. In this study, we evaluated the effect
of orally administered COX-1 and COX-2 inhibitors sulindac
and celecoxib on the promotion and progression stages
of UVB
photocarcinogenesis. To assess the anti-tumor promoting
effects of COX inhibitors, ninety mice were equally divided
into three groups. Group-I received no test agent, group-II
received 160ppm sulindac in drinking water and group-III
received 480 ppm celecoxib in the diet. Beginning fifteen
days after treatment with these agents, the animals were
exposed to UVB (180 mJ/ cm2, twice weekly) while continuing
on the agents for forty weeks. By week 14, 23% of the
UVB-treated Group-I mice developed tumors whereas only
10% of Group-III celecoxib-treated group developed tumors.
However, no tumors occurred in Group-II sulindac treated
mice. By week 18, 66% of Group-I mice but only 31% of
Group-III and Group-II treated mice developed tumors.
Tumor incidence reached 100% by week 29 in Group-I whereas
it was 83% and 66%
respectively in Group-III and Group-II mice respectively.
Similarly, the number of tumors/mouse at week 40 was 7.1
in Group-I whereas it was 4.7 and 3.0 in Groups III and
II. Both celecoxib and sulindac treatments resulted in
reduction of tumor size by 45% and 84% respectively. To
assess the effect of COX inhibitors on malignant progression
of UVB-induced tumors, mice were irradiated with 180 mJ/cm2
UVB (two times/week) for 28 weeks. At 28 weeks, ninety
animals with 5 or more tumors were withdrawn (no further
exposure to UVB) and divided into three groups. Group-1
mice (control) received water whereas group-2 mice received
celecoxib (480 ppm in diet) and group-3 received sulindac
(160 ppm in drinking water). Mice receiving celecoxib
and sulindac showed 33% and 55% fewer tumors and 40% and
60% reduction in tumor volume respectively compared to
untreated controls. These studies indicate that orally
administered COX inhibitors are potent anticancer agents
against UVB-induced photocarcinogenesis in SKH-1 mice.
HUMAN
RESEARCH
DNA
repair polymorphisms and liver cancer: effects on risk
and gene-environment and gene-gene interactions
Ruth M. Lunn, Douglas A. Bell, Xuguang Guo, Chien-Jen
Chen, Lian Wen Wang, Regina M. Santella, National Institute
of Environmental Health Sciences, Res. Triangle Park,
NC; Analytical Sciences, Inc., Research Triangle Park,
NC; National Taiwan University, Taipei, Taiwan; Mailman
School of Public Health, Columbia University, New York,
NY.
Hepatocellular
carcinoma (HCC) is the leading cancer in males and fifth
leading cancer in females in Taiwan. We evaluated the
effects of DNA repair polymorphisms (XRCC1 Arg194Trp,
XRCC1 Arg399Gln and XPD Lys751Gln) on HCC risk and their
ability to modulate exposures in a nested-case control
study. Cases and age-matched controls were identified
from a screening cohort of over 25,000 individuals from
Taiwan. Aflatoxin and HBV exposure had previously been
assessed by measuring aflatoxin-albumin adducts and HbsAg,
respectively, in serum. Genotyping for DNA repair polymorphism
was performed for 79 cases and 124 controls. Although
the XPD Lys751Gln polymorphism had no main effect on HCC
risk (OR=0.9, 95% CI=0.4 to 2.0), we found evidence to
suggest a gene (XPD)-environment (aflatoxin exposure)
interaction (p=0.06). The risk for HCC was highest among
aflatoxin exposed individuals with a Gln allele (OR=4.6,
95% CI=1.2 to 17.1). Stratified analyses by genotype showed
that HCC risk from aflatoxin exposure was approximately
six-fold higher in individuals with a 751Gln allele compared
to those with the 751 Lys/Lys genotype. The XPD polymorphism
did not appear to modify risks from HBV and smoking. We
also evaluated whether polymorphisms in the XRCC1 gene
affected HCC risk. The XRCC1 399Gln/Gln genotype was associated
with a modest increase in HCC risk (OR=3.3, 95% CI =0.9
to 12.5) and the combined XRCC1 194Arg/Trp and Trp/Trp
genotype was associated with a small reduction in risk
(OR =0.6, 95% CI= 0.3 to 1.1) albeit both associations
were not statistically significant. Lastly, we observed
a significant gene-gene interaction between the two XRCC1
polymorphisms (p=0.03) with the highest risk occurring
among 194Arg/399Gln carriers. Stratified analyses by each
polymorphism showed that the risk for a 399Gln allele
was elevated in the 194Arg/Arg strata but less than one
in the 194Trp allele strata. Similarly, the risk for the
194Arg/Arg genotype was elevated in the 399Gln allele
strata but less than one in the 399 Arg/Arg genotype strata.
Further studies with genotyping methods that detect haplotypes
may help interpret these results.
Relationship
between dietary carcinogens, DNA damage and
lobular neoplasia of the breast
Laverne A. Mooney, Kamala Maggard, Elizabeth Hovey, Freya
Schnabel, Deliang Tang, Mailman School Public Health,
New York, NY; NY Presbyterian Hospital, New York, NY;
Columbia Presbyterian Medical Center - Breast Service,
New York, NY.
Lobular
neoplasia (LN) of the breast represents a heterogenous
group of rare, noninvasive lesions incidentally found
in approximately 5% of breast biopsies. Primarily diagnosed
in premenopausal women, LN is associated with a 10-fold
increased risk of invasive breast cancer. To explore environmental
and genetic factors that might contribute to lobular neoplasia,
we assessed intake of dietary benzo(a)pyrene [B(a)P],
a specific PAH (e.g., from blackened, BBQ, and broiled
foods) and B(a)P-DNA damage in peripheral white blood
cells for patients from various risk groups. Patients
were recruited from NY Presbyterian Medical Center. Blood
samples and interview data (e.g., reproductive history,
dietary intake via the Block questionnaire and environmental
exposures) were collected. B(a)P-DNA adducts were measured
by HPLC. The 49 "healthy" women (without cancer) were
stratified into 5 breast cancer risk groups (4 according
to family history; 1 with LN determined by biopsy) by
a genetic counselor. Women (Mean age: 46 years (range:
29-70) were 84% Caucasian, 10% Hispanic, 6% Black/African
American. Religion: 48% Christian/Catholic; 38% Jewish;
2% Muslim; 12% Other. 90% were college graduates, 4/49
(8%) smoked in last 2 years (1-4 cig/day), and 63% reported
taking vitamin supplements. In all women B(a)P-DNA adducts
were associated with self-reported dietary intake of broiled,
smoked, charbroiled and fried meats (r=0.47, p=0.007)
in the last 2 weeks. The average number of servings was
5.0 (range 0-23). B(a)P-DNA adducts ranged from 0.125-
1.38 adducts/108 N. There was no association between adducts
and smoking, urban/rural residence, vitamins levels or
family history. None of the women with LN reported cigarette
smoking in the past 2 years. Women who had been diagnosed
with LN had significantly higher levels of B(a)P-DNA damage
(p<0.001) than women without a LN diagnosis (before
and after adjustment for dietary intake of broiled, smoked,
charbroiled and fried meats). LN patients were categorized
as either "higher" risk LN (LN and radial scar or atypical
ductal hyperplasia (ADH)) or "lower" risk LN (without
an additional high-risk lesion). A linear trend for B(a)P-DNA
adducts by LN status was observed (p=0.004) (see Table).
The OR for LN (either type) was 18.4 per unit of B(a)P-DNA
adducts, p=0.016. After adjustment for servings of charred
and broiled meats, the OR for LN was 20.6, p=0.016 per
unit of BP-DNA damage. These data indicate that environmental
exposure to common dietary carcinogens can be detected
at low levels, and the elevated adduct levels in women
with LN are suggestive of increased risk of invasive cancer
and genetic susceptibility.
B(a)P-DNA Adducts by Lobular Neoplasia Status
| Group |
B(a)P-DNA
Adducts/10(Mean +/- SD) |
Number |
P
value for trend* |
| No
LN Diagnosis |
0.33
+/- 0.31 |
30 |
0.004 |
| "Lower"
Risk LN |
0.54
+/- 0.61 |
4 |
|
| "Higher"
Risk LN |
0.90
+/- 0.22 |
3 |
|
*based
on ln transformed adduct levels.
Comparison
of biomarkers of prenatal carcinogenic exposure and DNA
damage in nonsmoking mothers and newborns in a multi-ethnic
population
Frederica P. Perera, Robin M. Whyatt, Virginia A. Rauh,
Robin S. Garfinkel, Dana L. Barr, John T. Bernert, Larry
L. Needham, Yanzhi Hsu, Deliang Tang, Center for Children's
Environmental Health, Mailman School of Public Health,
Columbia University, New York, NY; New York State Psychiatric
Institute, New York, NY;
Centers for Disease Control, Atlanta, GA.
The
purpose of the study was to assess susceptibility of the
fetus and ethnic differences in internal dose and biologic
response to common environmental carcinogens in the urban
environment. We have evaluated a battery of biomarkers
indicative of exposure and procarcinogenic damage within
an ongoing prospective cohort study of nonsmoking African-American
and Latina mothers and newborns in New York City. The
biomarkers (exposures) include cotinine measured by GC/MS
(environmental tobacco smoke); polycyclic aromatic hydrocarbon
(PAH)-DNA adducts by HPLC/fluorescence (PAH); and a series
of chlorinated organics and nonpersistent pesticides.
Comparing levels of several of these biomarkers in maternal
blood samples and umbilical cord blood collected at delivery,
concentrations of cotinine (219 pairs) were higher in
the newborns (0.27 ng/ml) than the mothers (0.15 ng/ml,
p<0.001). Adducts were also higher in the newborns
(0.25 vs. 0.19 per 108, p=0.09 (first 27 pairs), despite
the estimated 10-fold lower dose of PAH to the fetus compared
to the mother. Ethnic differences are being explored.
For example, restricting analysis to women who reported
household exposure to environmental tobacco smoke, cotinine
concentrations were higher in the African-American newborns
and mothers than the Latina mothers and newborns (p=0.05).
The data suggest differential susceptibility of the fetus
and possible ethnic differences in internal dose and potential
risk from certain environmental carcinogens.
NAT2
genotype, alcohol consumption and breast cancer development:
a novel gene-lifestyle interaction
Andrew G. Rundle, Deliang Tang, Laverne Mooney, Frederica
Perera, Mailman School of Public Health, New York, NY.
Epidemiologic
data have shown a link between alcohol consumption and
breast cancer. We have investigated this association further
by assessing possible interactions between alcohol consumption
and polymorphisms in phase II metabolic genes. Alcohol
induces many of the p450 phase I metabolic enzymes, and
ongoing induction of these enzymes would be expected to
generate increased levels of reactive metabolites of xenobiotics.
These reactive species are then further metabolized by
phase II enzymes, such as NAT2, with the net effect on
carcinogenicity depending on the substrate. Thus, polymorphisms
in phase II metabolic genes may interact with alcohol
consumption to influence breast cancer risk. We have conducted
a hospital based case-control study that enrolled 119
cases, 108 benign breast disease (BBD) controls and 141
healthy controls. Women were recruited from the clinic
and private practices of the Columbia-Presbyterian Medical
Center Breast Service prior to surgery and were assigned
to the case or BBD control group after the final diagnosis.
The BBD control group was restricted to women with benign
conditions without atypia. Healthy controls were recruited
from women receiving
regularly scheduled GYN checkups. The questionnaire collected
information on alcohol consumption and DNA samples from
white blood cells were analyzed for polymorphisms in the
NAT2 gene. Analyses showed that NAT2 genotype (fast/intermediate
vs. slow) acted as an effect modifier of the association
between alcohol consumption and breast cancer status,
such that there was a strong association between alcohol
consumption and breast cancer among NAT2 fast/intermediate
subjects but not among NAT2 slow subjects. When cases
were compared to healthy controls, among NAT2 fast/intermediate
subjects, the OR for current alcohol consumption was 4.11
(1.46-11.58) and among NAT2 slow subjects it was 0.83
(0.39-1.74) (p for interaction 0.01). When cases were
compared to BBD controls, among NAT2 fast/intermediate
subjects, the OR for current alcohol consumption was 4.70
(1.26-17.59) and among NAT2 slow subjects it was 1.34
(0.58-3.10) (p for interaction 0.21). Years of regular
alcohol consumption were strongly associated with case-healthy
control status among NAT2 fast/intermediate subjects but
not among NAT2 slow subjects (p for interaction = 0.04).
When cases were compared to BBD controls there was a similar,
but non-significant, association. The reduced effect seen
with BBD controls probably reflects a bias to the null
with this control group, due to the presence of shared
risk factors. These analyses controlled for age, ethnicity,
age at menarche, family history of breast cancer, age
at first birth, parity, and breast feeding history. This
work suggests that alcohol consumption is a risk factor
for breast cancer development only in a genetically susceptible
subgroup of women.
Polymorphisms
in the DNA repair enzyme XPD are associated with levels
of PAH-DNA adducts and breast cancer risk in a case-control
study
Deliang Tang, Stan Cho, Andrew Rundle, Senqing Chen, David
Phillips, Jingzi Zhou, Freya Schnabel, Alison Estabrook,
Frederica Perera, Columbia University, New York, NY; Institute
of Cancer Research, Sutton, UK; St. Luke's Hospital, New
York, NY.
We
present findings on the associations between DNA damage
from polycyclic aromatic hydrocarbons (PAH), risk of breast
cancer, and genetic susceptibility due to inherited polymorphisms
of the DNA repair enzyme XPD. Prior to surgery, breast
cancer cases and benign breast disease (BBD) controls
were enrolled into the study, took part in an interview
and donated a blood sample. A second control group of
healthy women recruited from the GYN practices were also
enrolled, took part in an interview and donated blood
samples. PAH-DNA adduct levels were measured by immunohistochemistry
in breast tissue samples retrieved from pathology blocks,
and by aromatic-DNA adducts were measured in mononuclear
white blood cells (MWBC) by 32P postlabelling. XPD genotype
at codons 312 and 751 was determined by PCR and RFLP analysis
using white blood cell DNA. The XPD analysis included
103 cases, 94 benign breast disease (BBD) controls, and
121 healthy controls. Neither of the polymorphisms were
associated with case-control status. However, XPD polymorphisms
at codons 312 and 751 were associated with higher levels
of PAH-DNA in tumor tissue from breast cancer cases. Subjects
with an AG or AA polymorphic genotype in codon 312 of
XPD had elevated levels of PAH-DNA adducts compared to
subjects with the GG genotype. PAH-DNA adducts were significantly
associated with increasing copy number
of the C allele for the codon 751 polymorphism (p for
trend, <0.01). Among subjects who had the polymorphic
XPD genotypes, adduct levels in tumor tissue were significantly
higher than in tissue from BBD controls. For both the
codon 312 and 751 polymorphisms, the associations between
the polymorphism and adduct levels was significantly different
in tumor tissue from benign tissue. Our results show XPD
polymorphisms are associated with increased levels of
DNA damage in tumor tissue, suggesting a possible basis
for genetic susceptibility to tumor progression.
Mammography
use among older breast cancer survivors
Julia E. Heck, Sherri Sheinfeld Gorin, Judith S. Jacobson,
Vijaya Sundararajan, Victor R. Grann, Alfred I. Neugut,
Department of Epidemiology, Mailman School of Public Health,
Columbia University, New York, NY; Herbert Irving Comprehensive
Cancer Center, Columbia University, New York, NY; Monash
University, Melbourne, Australia.
Breast
cancer survivors are at increased risk for a second malignancy,
especially for cancers of the contralateral breast, ovary,
uterus, and colon. We utilized the linked Surveillance,
Epidemiology, and End Results (SEER)-Medicare claims database
to compare 21,218 breast cancer survivors with 21,421
non-cancer controls on mammography use. Eligibility criteria
included age > 65 years and residence in a SEER area (comprising
about 14% of the US). Cases were women with an initial
diagnosis of breast cancer, stages 0-3, between 1990-1993
who had survived at least 24 months. Controls were a random
sample of Medicare beneficiaries who were frequency-matched
for age. We developed a multivariate regression model
of factors associated with use of screening, including
stage at diagnosis, race, and number of comorbidities.
Cases were 3.26 times as likely (95% CI: 3.17-3.35) as
controls to have received a mammogram in the 6 years after
diagnosis and treatment, controlling for age, race, stage
at diagnosis, and type of cancer treatment. Predictors
of mammography included white race, younger age, earlier
stage at diagnosis, having had a mastectomy, and 0-1 comorbidities.
The proportion of cases who had mammography decreased
over time, from 62.4% in the first 2 years after treatment
to 53.3% in years 5-6. The proportion of controls who
had mammography was significantly lower: 29.9% in years
1-2, dropping to 26.6% in years 5-6. In conclusion, breast
cancer survivors are much more likely than controls to
receive mammography. This association is consistent with
their elevated risk for a new primary breast cancer. Supported
by The Avon Products Foundation.
Molecular
epidemiology can be a valuable tool in cancer prevention
Frederica P. Perera, Mailman School of Public Health,
Columbia University, New York, NY.
A
major stumbling block in cancer prevention has been the
lack of "early warning systems" to identify risk factors
as well as populations and individuals at greatest risk.
In 1982 molecular epidemiology was proposed as a new preventive
research approach in which "advanced laboratory methods
are used in combination with analytic epidemiology to
identify at the biochemical or molecular level specific
exogenous and/or host factors that play a role in human
cancer causation" (1). The concept was that, by introducing
biomarkers of dose, effect, and susceptibility into epidemiology,
researchers "should be able to predict human risks more
precisely than hitherto possible"; this knowledge could
then provide the basis for interventions (1). The field
has developed rapidly since the 1980s and has demonstrated
considerable potential in reducing the burden of human
cancer (2-9).
In
recent decades molecular epidemiology has provided new
insights into the important roles of environmental exposures
and susceptibility factors in human cancer—with clear
implications for prevention (see ref. 9 for review). The
exposures include tobacco smoke, polycyclic aromatic hydrocarbons
(PAH) and other aromatic carcinogens, aflatoxin B1, hepatitis
B virus, and benzene which are known or suspected risk
factors in lung, breast, and liver cancer and leukemia.
The biomarkers utilized in this research include carcinogen-DNA
adducts (markers of biologically effective dose), hypoxanthine-guanine
phosphoribosyltransferase (HPRT) and P53 mutation, activation
of oncogenes, and chromosomal aberrations (markers of
preclinical effect), as well as common polymorphisms in
metabolic or repair genes (e.g. GST, CYP, NAT, XRCC1)
and
plasma levels of antioxidants (markers of susceptibility).
The specific contributions of molecular epidemiology have
been in four areas: 1) providing new evidence that preventable
environmental agents pose carcinogenic risks, 2) helping
establish the
causal roles of environmental factors in cancer, 3) identifying
environment-susceptibility interactions and populations
at greatest risk, and 4) developing new intervention strategies.
Of
particular importance, molecular epidemiologic data indicate
that risk assessment and intervention strategies should
focus on subgroups at elevated risk because of the combination
of exposure and genetic or acquired susceptibilities,
including young age. For example, we have found that there
is considerable variability in the concentration of aromatic
carcinogen-DNA adducts present in white blood cells (WBC)
of healthy smokers, after adjusting for the amount the
individuals smoked (10). Smokers with elevated levels
of aromatic DNA adducts in their WBCs were approximately
three
times more likely to be diagnosed with lung cancer 1-13
years later than smokers with lower adduct concentrations
(odds ratio, 2.98; 95% confidence interval, 1.05-8.42;
P = 0.04). Overall, lung cancer risk was elevated among
individuals with the GSTM1 null/P1 Val genotype (Perera
et al., submitted). The findings provide direct evidence
that individuals who become cases have greater biological
susceptibility to PAH/aromatic and other carcinogens.
Ambient
air pollution is another complex mixture containing aromatic
combustion by-products. Experimental and human evidence
indicates that the fetus and young child have greater
vulnerability to many carcinogens due to the combination
of biologic factors and the long lifetime over which cancer
initiated early can develop. We therefore compared biomarkers
in 160 maternal and newborn umbilical cord blood samples
obtained at delivery, and found that the levels of aromatic-DNA
adducts were significantly higher in the newborns (P=0.002)
despite the fact that the estimated transplacental dose
of the carcinogens is only one-tenth that to the mother
(11). In the newborns, aromatic adducts were significantly
associated with somatic gene mutations as measured by
HPRT mutant frequency (Mf) (P=0.03) (Perera et al., submitted).
The association remained after controlling for maternal
exposure to tobacco smoke. This study indicates differential
susceptibility of the fetus and provides a molecular link
between transplacental exposure to aromatic compounds
commonly present in ambient air pollution and somatic
mutation in newborns, a finding that is directly relevant
to risk of cancer. Similarly, we have previously reported
a significant relationship between WBC aromatic-DNA adducts
and chromosomal aberrations in adults, after controlling
for cigarette smoking and age (P=0.01) (12). Like adducts,
chromosomal aberrations have been prospectively
shown to be predictive of subsequent cancer (13).
Research
results are increasingly being translated into behavioral,
chemopreventive and regulatory interventions aimed at
protecting "at risk populations". However, there remain
gaps in knowledge. To fill these gaps, the secon
