The Toxicologist - Society of Toxicology

ole in invasion and metastasis of cancer cells and are markers of malignant transformation
by arsenic or Cd. In SCs trans-cultured with CAsE-PE or CTPE cells,
MMP transcript increased after only 1 week of trans-culture, and secreted MMP-2
and MMP-9 activity increased after 2 weeks. PTEN, a tumor suppressor gene with
a critical role in SCs differentiation, is often inactivated in malignancies. PTEN
transcript was highly suppressed after only 1 week of trans-culture of SCs with either
CAsE-PE or CTPE cells. K5 and P63 are markers of prostate basal cells, the
absence of which is used for pathological diagnosis of prostate cancer. K5 and P63
were also markedly suppressed by trans-culture of SCs with CAsE-PE or CTPE
cells. Therefore, arsenic- and Cd-induced malignantly transformed prostate epithelia
impacts nearby normal SCs, apparently driving them towards a malignant phenotype
while not actually having physical contact.
1993 OVERABUNDANCE OF PUTATIVE CANCER STEM
CELLS IN HUMAN SKIN KERATINOCYTE CELLS
MALIGNANTLY TRANSFORMED BY ARSENIC.
Y. Sun, E. Tokar and M. Waalkes. National Toxicology Program, NIEHS, Research
Triangle Park, NC.
Arsenic is a human skin carcinogen. Cancer is probably a disease driven by stem
cells (SCs) and SCs are likely a key target during arsenic oncogenesis. In utero arsenic
exposure predisposes mice to skin carcinoma formation with an overproduction
of cancer SCs (CSCs) and distortion of CSC signaling and population dynamics.
Therefore, here we hypothesized that accumulation of CSCs may occur in a
human skin keratinocyte line during arsenic-induced malignant transformation in
vitro. Thus, the HaCaT cell line, which was malignantly transformed by sodium arsenite
(100 nM, 30 weeks; termed As-TM cells) in prior work, was further studied
for the quantity and nature of SC/CSCs during transformation. SCs/CSCs were
isolated from As-TM cells and control cells by using a positive magnetic bead isolation
system that purifies CD34 positive cells. CD34 is a cell surface marker for
both human skin SCs and CSCs. Using this system, there were 2.5 times more
SCs/CSCs isolated from As-TM cells than from control cells. Holoclones are dense,
tightly packed clusters of cells formed in culture by SCs/CSCs and enriched in
these same cells. After isolation of SC/CSCs, holoclone production from CSCs isolated
from As-TM cells was 2.5-fold higher at 1 week and 3.5-fold higher at 2
weeks than SCs isolated from control cells. SC characteristics and potential malignant
phenotype were assessed in SC/CSCs isolated from control and As-TM cells
and compared to their parental lines. Transcript level of SC/CSC markers were elevated
in both As-TM CSCs and control SCs compared to parental cells, but CSCs
from As-TM cells had elevated Oct-4, K5, K15 transcripts, and dramatically
stronger staining for p63, Notch1, K19 compared to control SCs. As-TM CSCs
also showed markedly elevated MMP-9 secretion and colony formation in agar,
both indicators of cancer phenotype, even when compared to total population of
As-TM cells. Thus, arsenic-induced malignant phenotype is particularly pronounced
in SCs during transformation of human skin cells and occurs concurrently
with an overproduction of CSCs.
1994 TRANSFORMATION OF NORMAL HUMAN BREAST
EPITHELIAL (MCF-10A) CELLS BY ARSENITE AND
CADMIUM INDUCES THE OVER-EXPRESSION OF
NEURONAL SPECIFIC ENOLASE (ENOLASE-2).
M. Soh, S. H. Garrett, C. Bathula, D. A. Sens, S. Somji and M. Sens. Pathology,
University of North Dakota, Grand Forks, ND.
Arsenite (As +3 ) and cadmium (Cd +2 ) exposure have been associated with many
types of cancers including bladder, skin, liver, prostate and lung, and in case of
breast, they have been implicated in estrogen receptor mediated gene transcription,
endocrine disruption, DNA damage and altered cell growth. Previous studies from
our laboratory haves shown that both As +3 and Cd +2 can directly malignantly transform
the non-tumorigenic urothelial cell line UROtsa. The goal of this study was
to determine if both these heavy metals could also transform the breast epithelial
cell line MCF-10A. For this purpose the MCF-10A cells were exposed to 1 μM
As +3 or Cd +2 for a long period of time with the end point being the ability to form
colonies in soft agar. It was the demonstrated that long term exposure resulted in
selection of cells that were able to form colonies in soft agar. These transformed
cells were subjected to microarray analysis and one of the differentially expressed
gene that was identified was enolase-2 (ENO-2) which is a glycolytic enzyme that
catalyses the conversion of 2-phosphoglycerate to phosphoenol pyruvate. ENO-2
has shown to be over expressed in neuronal and neuro-endocrine tumors. It is also
over expressed in certain breast cancers that have a neuro-endocrine differentiation.
The next goal of the study was to determine if Cd +2 and As +3 could induce the expression
of this enzyme in the MCF-10A cells upon short and long term exposure.
The results obtained indicate that both the metals can up-regulate the expression of
ENO-2 in a time and dose dependent manner. Although the mechanism involved
in the up-regulation of ENO-2 is unknown, our study provides evidence that the
increased expression of ENO-2 may have implications in the increased glycolytic
capacity of tumors
1995 SPARC GENE EXPRESSION IS REPRESSED IN HUMAN
UROTHELIAL CELLS (UROTSA) EXPOSED TO OR
MALIGNANTLY TRANSFORMED BY CADMIUM OR
ARSENITE.
J. L. Larson 1 , S. Somji 2 , D. A. Sens 2 , M. Sens 2 , S. H. Garrett 2 and J. R.
Dunlevy 1 . 1 Anatomy and Cell Biology, University of North Dakota, Grand Forks,
ND and 2 Pathology, University of North Dakota, Grand Forks, ND.
SPARC belongs to a class of extracellular matrix-associated proteins that have counteradhesive
properties. The ability of SPARC to modulate cell-cell and cell-matrix
interactions provides a strong rationale for studies designed to determine its expression
in cancer. The objective of this study was to determine if SPARC expression
was altered in cadmium (Cd +2 ) and arsenite (As +3 ) induced bladder cancer and if
these alterations were present in archival specimens of human bladder cancer. The
expression of SPARC was determined in human parental UROtsa cells, their Cd +2
and As +3 transformed counterparts and derived tumors, and in archival specimens
of human bladder cancer using a combination of real time reverse transcriptase
polymerase chain reaction, western blotting, immunofluoresence localization and
immunohistochemical staining. It was demonstrated that SPARC expression was
down-regulated in Cd +2 and As +3 transformed UROtsa cells. In addition, the malignant
epithelial component of tumors derived from these cell lines were also
down-regulated for SPARC expression, but the stromal cells recruited to these tumors
was highly reactive for SPARC. This finding was shown to translate to specimens
of human bladder cancer where tumor cells were SPARC negative, but stromal
cells were positive. Acute exposure of UROtsa cells to both cadmium and
arsenite reduced the expression of SPARC through a mechanism that did not involve
changes in DNA methylation or histone acetylation. These studies suggest
that environmental exposure to As +3 or Cd +2 can alter cell-cell and cell-matrix interactions
in normal urothelial cells through a reduction in the expression of SPARC.
The SPARC associated loss of cell-cell and cell-matrix contacts may participate in
the multi-step process of bladder carcinogenesis.
1996 DIFFERENCES IN THE EPIGENETIC REGULATION OF
METALLOTHIONEIN-3 GENE EXPRESSION BETWEEN
PARENTAL AND CADMIUM- OR ARSENITE-
TRANSFORMED HUMAN UROTHELIAL CELLS.
D. A. Sens, S. H. Garrett, A. Ajjimaporn, M. Sens and S. Somji. Pathology,
University of North Dakota, Grand Forks, ND.
Cadmium (Cd +2 ) and arsenite (As +3 ) and are human carcinogens and exposure to
them has been associated with the development of bladder cancer. Previous work
from our laboratory has shown that the UROtsa cell line can be directly malignantly
transformed by both Cd +2 and As +3 and that the resulting tumors retain features
of urothelial cancer. These transformed cell lines in cell culture do not express
metallothionein-3 (MT-3); despite the fact that most human bladder tumors overexpress
this gene. However, when they are transplanted as tumors they show strong
expression of MT-3. This difference in MT-3 expression suggests that the mechanism
of MT-3 gene silencing between the parental and transformed urothelial cells
are different. The goal of this study was to determine if epigenetic modifications
control urothelial MT-3 gene expression and if regulation is altered by malignant
transformation by Cd +2 or As +3 . For this purpose, the parent and the transformed
cell lines were exposed to the histone deacetylase inhibitor MS-275 and RNA was
isolated and subjected to RT-PCR. The results obtained showed that MS-275 induced
MT-3 mRNA expression in both parental UROtsa cells and their transformed
counterparts. Chromatin Immunoprecipitation analysis showed that the
metal-responsive transcription factor-1 (MTF-1) binding to the metal response elements
(MRE) of the MT-3 promoter was restricted in parental cells under basal
conditions, but MTF-1 binding to the MREs was unrestricted in the transformed
cell lines. Histone acetylation of H4 and trimethylation of H3K4, H3K9 and
H3K27 were compared between the parental and transformed cells. These studies
showed that there is a fundamental difference in the accessibility of the MRE elements
to MTF-1 binding within the MT-3 promoter between the parental UROtsa
SOT 2011 ANNUAL MEETING 427