Evidence for a Zinc Uptake Transporter in Human Prostate Cancer
Cells Which Is Regulated by Prolactin and Testosterone*
Leslie C.
Costello
,
Yiyan
Liu,
Jing
Zou, and
Renty B.
Franklin
From the Cellular and Molecular Biology Section, Department of Oral
and Craniofacial Biological Sciences, University of Maryland Dental
School, Baltimore, Maryland 21201
 |
ABSTRACT |
The glandular epithelial cells of the human
prostate gland have the unique capability and function of accumulating
the highest zinc levels of any soft tissue in the body. Zinc
accumulation in the prostate is regulated by prolactin and
testosterone; however, little information is available concerning the
mechanisms associated with zinc accumulation and its regulation in
prostate epithelial cells. In the present studies the uptake and
accumulation of zinc were determined in the human malignant prostate
cell lines LNCaP and PC-3. The results demonstrate that LNCaP cells and
PC-3 cells possess the unique capability of accumulating high levels of
zinc. Zinc accumulation in both cell types is stimulated by
physiological concentrations of prolactin and testosterone. The studies
reveal that these cells contain a rapid zinc uptake process indicative of a plasma membrane zinc transporter. Initial kinetic studies demonstrate that the rapid uptake of zinc is effective under
physiological conditions that reflect the total and mobile zinc levels
in circulation. Correspondingly, genetic studies demonstrate the
expression of a ZIP family zinc uptake transporter in both LNCaP and
PC-3 cells. The rapid zinc uptake transport process is stimulated by
treatment of cells with physiological levels of prolactin and
testosterone, which possibly is the result of the regulation of the
ZIP-type zinc transporter gene. These zinc-accumulating
characteristics are specific for prostate cells. The studies support
the concept that these prostate cells express a unique
hormone-responsive, plasma membrane-associated, rapid zinc uptake
transporter gene associated with their unique ability to accumulate
high zinc levels.
 |
INTRODUCTION |
Zinc is an essential component of all cells. It is required for a
variety of cellular activities such as metalloenzyme activity, nucleoprotein and nucleic acid structure, and transcription factor interactions. Typically, intracellular zinc is found predominantly (> 95%) bound to high molecular weight ligands such as metalloenzymes, metalloproteins, nucleoproteins, and nucleic acids. Very little zinc is
available as free or loosely bound zinc, which we will refer to as " mobile reactive zinc" (for review, see Refs. 1-3).
The prostate gland of humans and other animals is unique in that it
accumulates much higher zinc levels than any other soft tissues in the
body. For detailed and extensive reviews of zinc-citrate relationships
in prostate, see Refs. 4-6. The special functions associated with the
high zinc level of the prostate have not been resolved. The ability of
the prostate to accumulate high zinc levels is a function of the
glandular secretory epithelial cells. Our recent studies (7) with rat
prostate lobes have demonstrated that the epithelial cells contain high
levels of intracellular zinc, and, most importantly, contain high
levels of mitochondrial zinc. The accumulation of zinc results in the
inhibition of mitochondrial aconitase activity which minimizes the
ability of these cells to oxidize citrate (8). This is an important
relationship associated with the unique functional and metabolic
capability of the prostate to accumulate high citrate levels. This
results in a significant portion (about 30%) of the total zinc of
citrate-producing prostate cells existing in a chelated form with
citrate (9-11), which is in contrast to other cells in which greater
than 95% of the intracellular zinc is bound to macromolecules in an
immobile form.
Animal studies have revealed that the accumulation of zinc in the
prostate is regulated by testosterone and prolactin (6, 7, 12). In
rats, both hormones increase the cellular and mitochondrial levels of
zinc in lateral prostate cells; both hormones decrease the cellular and
mitochondrial levels of zinc in ventral prostate cells; and neither
hormone has any effect on the zinc levels of dorsal prostate cells or
non-prostate cells (7). Dorsal prostate and non-prostate cells are not
citrate-producing cells, whereas lateral and ventral prostate cells are
citrate-producing cells. This raised the important question as to which
types of epithelial cell exist in the human prostate in relation to its function of producing, accumulating, and secreting extremely high levels of zinc. Moreover, it is well established that naturally occurring malignant prostate cells lose the ability to accumulate zinc.
Despite these important functional and pathological relationships, virtually no information exists concerning the mechanism(s) involved in
the accumulation of zinc and its regulation in human prostate epithelial cells. The present report reveals that 1) LNCaP and PC-3
cells (human malignant prostate cell lines) exhibit the capability of
accumulating high zinc levels; 2) a rapid zinc uptake transport mechanism is associated with the accumulation of high zinc levels; 3)
prolactin and testosterone are positive regulators of the transport mechanism; and 4) LNCaP and PC-3 cells express a hormonally regulated ZIP-type plasma membrane zinc uptake transporter.
 |
EXPERIMENTAL PROCEDURES |
For these studies we elected to employ the human malignant cell
lines LNCaP and PC-3. As presented under "Results," both cell lines
exhibit the capability of accumulating high zinc levels under the
in vitro conditions employed; therefore, they are excellent models for studying the mechanisms and regulation of zinc accumulation. Moreover, LNCaP cells exhibit the characteristics of citrate-producing cells with a limiting m-aconitase, whereas PC-3 cells are
citrate-oxidizing cells in which m-aconitase is not limiting
(13). In addition, LNCaP cells have a very low tumorigenicity compared
with the highly tumorigenic PC-3 cells. Thus, comparative studies in
these cell types will offer the future advantage of establishing the
zinc relationships to citrate metabolism and malignancy.
The conditions for culture and harvesting of the cells were the same as
described previously (13). The culture medium was RPMI 1640 and 10%
fetal bovine serum (FBS).1
Generally, at 18 h before cell harvesting, the medium was changed to RPMI 1640 without the addition of FBS to minimize any effect of
unidentified components of FBS which might influence the accumulation of zinc. The harvested cells were washed and suspended in Hanks' balanced salt solution (HBSS) and used in the experiments. Transfection of PC-3 cells with androgen receptor was achieved as described previously (14).
For experiments involving total zinc accumulation, the incubation
medium was HBSS. Depending upon the experiment, zinc and hormones were
added to the medium. After an appropriate incubation period at
37 °C, the cells were quickly centrifuged and washed with HBSS. The
washed cells were then digested and prepared for atomic absorption
assay of zinc as described previously (7). The cellular zinc levels
were calculated as ng/mg cell protein. Cell protein was assayed by the
method of Bradford (15).
Zinc transport was determined by 65Zn rapid uptake by the
cells. In these experiments the harvested cells (2-5 × 106) were added to microcentrifuge tubes. Generally, 200 µl of HBSS containing 65ZnCl2 was added to
the microcentrifuge tubes. After the appropriate incubation period, 1.5 ml of cold stop solution (250 mM sucrose containing 1 mM EDTA and 50 mM Hepes buffer, pH = 7.2)
was rapidly added to the microcentrifuge tubes followed by rapid
centrifugation. The packed cells were rapidly resuspended in 1.5 ml of
cold stop solution and centrifuged. The packed cells were collected in
liquid scintillation mixture and 65Zn counted in a liquid
scintillation counter.
The following procedures were employed to determine the expression of a
plasma membrane zinc uptake transporter in the prostate cells. A ZIP
family of metal transporters which includes zinc transporters has been
identified in plant and yeast cells (16-19). A putative mammalian zinc
transporter (H2O615) has been identified in the Expressed Sequence Tags
data base which shares remarkable homology with the
Arabidopsis ZIP1. ZIP1 is a plasma membrane zinc uptake
transporter, and therefore was considered by us to be a prime candidate
for expression in prostate cells. The partial cDNA clone (H2O615)
was used to screen the Human Universal cDNA Library Array (HUCL)
from the Stratagene Corporation. We identified a cDNA clone in the
HUCL which hybridized, under high stringency conditions, with the EST
partial clone. We designated this HUCL clone "hZIP1"
(human ZIP1) which was used to determine the expression of
the putative zinc uptake transporter in LNCaP and PC-3 cells. The
procedures for RNA extraction and Northern blot analysis for the
detection of hZIP1 mRNA have been described (20).
The zinc accumulation and kinetic experiments were repeated two or more
times to ensure the reproducibility of the results, and all assays were
run in duplicate or triplicate depending upon the volume of sample
available for assay. Without exception, the standard errors within each
experiment were less than 5% of the mean values presented under
"Results." Data presented as statistically significant represent a
p < 0.05, although most of the statistically significant differences exhibited p < 0.01 values. The
reproducibility and consistency of the results are demonstrated by the
fact that no repeated experiment deviated from the typical effects
presented under "Results."
 |
RESULTS |
In the first series of studies we had to determine if LNCaP cells
and/or PC-3 cells exhibited the capability of accumulating high zinc
levels, which would be representative of the unique zinc-accumulating
characteristic of the human prostate. The endogenous level of cellular
zinc in LNCaP and PC-3 cells and the accumulation of zinc in cells
exposed to zinc added to the medium were established (Table
I). The "endogenous " level is
defined as the concentration in the cells grown and maintained in
unsupplemented medium with respect to zinc. We assayed the media and
found that RPMI/FBS contains about 0.05 µg zinc/ml; when FBS is
omitted, the RPMI medium as well as HBSS contains no detectable zinc.
The mean endogenous level of zinc in LNCaP cells (248 ng/mg protein)
was 51% higher than the level (164 ng/mg protein) in PC-3 cells.
Throughout these studies and without exception the endogenous cellular
zinc concentration of LNCaP cells was consistently higher than the
corresponding zinc level of PC-3 cells. Consequently it is clear that
LNCaP cells contain a significantly higher endogenous zinc level than PC-3 cells. When the harvested cells were incubated for 4 h in HBSS medium supplemented with 1 µg/ml zinc (the approximate
concentration in plasma) in the form of zinc sulfate, the cellular
level of zinc in LNCaP increased by 98% to 493 ng/mg protein; and PC-3 zinc levels increased 118% to 358 ng/mg protein. Thus, both LNCaP and
PC-3 cells took up and accumulated zinc from the medium; however, LNCaP
cells maintained significantly higher (37%) zinc levels than PC-3
cells in the presence of physiological exogenous zinc levels. In
experiment 3 (Table I) we had also included (not shown) zinc
measurements in squamous carcinoma cells to represent a non-prostate cell type. The zinc level of the squamous carcinoma cells was 174 ng/mg
protein compared with 428 and 306 for LNCaP and PC-3 cells,
respectively. Thus, the zinc level of the non-prostate cells was
significantly less than the prostate cells. This correlates with the
rat studies in which non-prostate cells (kidney and liver cells)
contain significantly lower zinc levels than prostate cells (7).
View this table:
[in this window]
[in a new window]
|
Table I
Total cellular zinc levels of LNCaP and PC-3 cells
The cells were grown in RPMI 1640 and 10% FBS. The medium was changed
to RPMI 1640 at 18 h before harvesting. The harvested cells were
incubated for 4 h in HBSS ( zinc) or HBSS containing 1 µg
zinc/ml (+zinc). The values are the means (±S.E.) expressed as ng of
zinc/mg of cell protein.
|
|
We then determined the effects of prolactin and testosterone treatment
of the cells on zinc accumulation (Table
II). In these experiments the harvested
cells were incubated for 4 h in HBSS medium containing 1 µg/ml
zinc and 30 nM prolactin, 10 nM testosterone, or vehicle (control). With LNCaP cells, zinc accumulation was increased
significantly by both testosterone (+51%) and prolactin (+37%). With
PC-3 cells, zinc accumulation was increased significantly by prolactin
(+40%), but testosterone had no significant effect. The absence of an
effect of testosterone on PC-3 cells might be expected because these
cells do not contain androgen receptor; therefore we determined the
effect of testosterone on PC-3 cells that were transfected with
androgen receptor (Table III).
Testosterone treatment increased the accumulation of zinc in the
transfected cells by 32% but had no effect on the nontransfected PC-3
cells. Moreover, the zinc levels of untreated transfected and
nontransfected cells were the same, so transfection alone had no effect
on zinc accumulation. The transfection result is also important because it indicates that the testosterone effect is mediated via a
gene-regulated mechanism. Consequently the effect of actinomycin on the
hormonal stimulation of zinc accumulation was determined (Table II).
With LNCaP cells actinomycin completely inhibited the stimulatory
effect of both prolactin and testosterone. With PC-3 cells actinomycin inhibited the effect of prolactin. Because the PC-3 cells were not
transfected, testosterone had no effect on zinc accumulation, and
actinomycin had no effect. The results indicate that the stimulatory effects of prolactin and testosterone on zinc accumulation involve some
aspect of gene expression. Neither testosterone nor prolactin treatment
had any effect on the zinc levels of squamous carcinoma cells cells
(results not shown); therefore the hormonal effects on the prostate
cells exhibit cell specificity.
View this table:
[in this window]
[in a new window]
|
Table II
Effects of prolactin and testosterone on zinc accumulation in LNCaP and
PC-3 cells
Cells were incubated in HBSS containing 1 µg/ml zinc plus 30 nM prolactin or 10 nM testosterone or vehicle
(control), with or without 8 µM actinomycin. The values
are the means (±S.E.) reported as ng of zinc/mg of protein. Cont.,
control; +Test., testosterone added; Act., actinomycin; + PRL,
prolactin added.
|
|
View this table:
[in this window]
[in a new window]
|
Table III
Effect of androgen receptor transfection on testosterone stimulation of
zinc accumulation in PC-3 cells
The conditions are the same as presented in Table II. The values are
the means (±S.E.) expressed as ng of zinc/mg of protein. [% change
from control.]
|
|
The protocol for the experiments described above involved the
simultaneous incubation of harvested cells for 4 h in medium containing 1 µg/ml zinc and hormone; however it was now important to
establish how early the cellular accumulation of zinc would be
manifested. To eliminate the hormone induction period from the zinc
uptake kinetics, PC-3 cells were pretreated with prolactin-supplemented RPMI medium overnight to maximize the prolactin effect compared with
cells maintained in prolactin-free RPMI. The treated and control cells
were harvested, washed, and incubated in HBSS containing 1 µg/ml zinc
over a period of 60 min. The results (Fig.
1) demonstrate that a prolactin-induced
increase in zinc accumulation was clearly evident by 60 min. Of special
importance is the magnitude of the difference in zinc accumulation by
60 min associated with the hormone effect, a difference of 109 ng/mg
protein, which is a 33% increase in total zinc. This is important
because less than 5% of the total cellular zinc of mammalian cells is
represented by mobile (free or loosely bound) reactive zinc, which
would calculate to approximately 20 ng/mg protein. Thus, the difference
of 109 ng/mg protein would actually represent an increase of 5-fold in relation to mobile zinc. Obviously, the magnitude of this increase would require that the increase in zinc accumulation must have been
initiated very early, probably within minutes, in order for this
accumulated difference to be so large by 60 min.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of prolactin pretreatment of PC-3
cells on the accumulation of zinc. The cells were exposed for
18 h to RPMI medium containing either 30 nM prolactin
or vehicle (minus prolactin). The pretreated cells were harvested and
incubated in HBSS containing 1 µg/ml zinc for periods up to 60 min.
|
|
The above results indicated that the accumulation of zinc involved a
rapid zinc uptake process. To determine this possibility we determined
the rapid uptake of 65Zn in PC-3 cells. The results (Fig.
2) demonstrate the existence of a rapid
uptake mechanism. The zinc uptake rate was greatest within the first
30 s, and the rate declined thereafter. If this rapid uptake
process were linked to the zinc accumulation in these cells, we
reasoned that the rapid uptake of zinc should exist in both cell types
and should be hormone-responsive as is the case with zinc accumulation.
In these studies, the harvested cells were divided into two groups: a
group that was pretreated by incubation for 3 h in medium
containing 30 nM prolactin versus a control group containing hormone vehicle. The results (Fig.
3) demonstrate that LNCaP cells also
exhibited the existence of a rapid zinc transport mechanism; however,
the rapid uptake rate of LNCaP cells was 3-5 times greater than PC-3
cells, which is consistent with higher zinc accumulation in LNCaP cells
compared with PC-3 cells. Prolactin pretreatment markedly stimulated
the rapid uptake of zinc by LNCaP and PC-3 cells. The effect is more
pronounced in LNCaP cells, but in both cases the hormonal increase was
about 2-fold. Moreover, the effect of the hormone appeared to involve an increase in the maximal rate of zinc uptake. This too is indicative of an effect on a zinc transporter. The prolactin-induced effect within
a 3-h exposure time to prolactin is the same time frame involved in
maximal prolactin stimulation of other gene-regulated metabolic
activities of prostate cells (4-6).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2.
Rapid uptake of zinc by PC-3 cells. PC-3
cells were incubated in HBSS containing 0.5 µg/ml zinc labeled with
65Zn.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of prolactin pretreatment of LNCaP and
PC-3 cells on the rapid uptake of zinc. Cells were pretreated for
3 h with HBSS containing 30 nM prolactin, 10 nM testosterone, or vehicle. The pretreated cells were
harvested and incubated in HBSS medium containing 1 µg/ml zinc
labeled with 65Zn.
|
|
In plasma the total zinc concentration is about 1 µg/ml. Of this
total, about 34% is firmly bound (mainly to globulin), and 66% is
loosely bound, which represents the "mobile" component available
for cellular uptake; therefore the rapid uptake of zinc by LNCaP and
PC-3 cells over the range of 0.1-2.0 µg/ml zinc was determined (Fig.
4). Rapid zinc uptake at 25 °C by
LNCaP and PC-3 cells was increased as the extracellular concentration
of zinc was increased up to approximately 1.0 µg of zinc/ml. The
maximal uptake rate for both cells occurred at about 1.5 µg of
zinc/ml. It is also apparent that the rapid uptake process was
saturable, which is representative of either an active or facilitative
transport process. The rapid transport rates at zinc levels
representative of circulating levels of mobile zinc are about three
times greater for LNCaP than PC-3 cells. This is consistent with the
results described above and is consistent with the capability of LNCaP cells to accumulate higher zinc levels than PC-3 cells. Because of the
low 65Zn uptake rates at this low physiological zinc
concentration range, we established the diffusion of zinc into the
cells. For this we compared the 65Zn rapid uptake of PC-3
cells at 4 and 25 °C (Fig. 4). At 4 °C, the rapid zinc uptake
should be the result mainly of the diffusion of zinc into the cells
with the zinc uptake by transport being minimal. This is evident in
that zinc uptake at 4 °C was linear over the zinc concentration
range and exhibited no saturation kinetics. It is readily apparent that
the influx of zinc at 4 °C over this physiological range of was
minimal in relation to the uptake of zinc at 25 °C. Even at
extremely low zinc concentrations (e.g. 0.15-0.3 µg/ml)
the rapid transport of zinc into the cells was evident. At the expected
normal circulating range of diffusable zinc (0.3-0.9 µg/ml), the
cellular uptake of zinc by transport was increased over the diffusion
of zinc by 3-fold for PC-3 cells and by 9-fold for LNCaP cells.
Collectively these studies demonstrate the existence of a rapid zinc
uptake transporter in the prostate cells which is capable of
functioning at zinc levels representative of plasma zinc
concentrations. We are now conducting kinetic studies to establish the
optimal conditions for this rapid zinc transport at which time we will
be able to determine accurately the Km and
Vmax for this transporter and the effects of
hormonal stimulation on the kinetics.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Rapid uptake of zinc by LNCaP and PC-3 cells
at physiological levels of zinc. The uptake rates at 4 °C
reflect mainly the net diffusion of zinc into the cells. The difference
in zinc uptake at 4 °C and at 25 °C reflects the rapid transport
of zinc into the cells.
|
|
In all of the hormonal studies described above, 10 nM
testosterone and 30 nM prolactin were employed, which
proved effective to stimulate zinc uptake in the prostate cells. The
normal circulating levels of prolactin and testosterone are
approximately 2 nM and 1 nM, respectively. It
was important to determine if lower hormonal concentrations were
effective in stimulating zinc uptake. Table IV demonstrates that prolactin as low as
0.6 nM significantly stimulated rapid zinc uptake of LNCaP
cells, and a dose response occurred as the concentration of prolactin
was increased. Testosterone at a concentration of 0.1 nM
significantly increased the rapid zinc uptake, and a dose response
existed. With PC-3 cells, 0.6 nM prolactin also stimulated
rapid zinc uptake, while testosterone had no effect as would be
expected in these androgen receptor-deficient cells. It is of interest
to note that prolactin had a more pronounced effect on stimulation of
the rapid uptake of zinc in PC-3 cells compared with LNCaP cells,
although LNCaP cells accumulate higher zinc levels. This might be
because of the possibility that the rapid transport mechanism is more
active in the LNCaP cells in the absence of added hormone, which
contributes to the higher endogenous zinc levels of these cells.
Nevertheless, these studies demonstrate that both prolactin and
testosterone at low physiological levels are effective regulators of
zinc uptake in prostate cells. This is consistent with the rat studies
in which the combination of in vivo and in vitro
experiments demonstrated that prolactin and testosterone are
physiological regulators of zinc accumulation in prostate (7).
View this table:
[in this window]
[in a new window]
|
Table IV
Effects of varying concentrations of prolactin and testosterone on the
rapid uptake of zinc by LNCaP and PC-3 cells
Cells were pretreated for 3 h with hormone or vehicle followed by
incubation for 2 min in HBSS containing 65Zn at a zinc
concentration of 1 µg/ml. The uptake rate is ng of zinc/million
cells/2 min. The values are the means (±S.E.) and percent increase
versus control (no hormone).
|
|
The studies described above provided compelling kinetic evidence for
the existence of a zinc uptake transporter in prostate cells. The
hormonal stimulation and its inhibition by actinomycin of zinc uptake
led us to consider that the prostate cells likely expressed a rapid
uptake transporter gene. A putative plasma membrane zinc transporter in
humans has been identified based on the high homology to the
Arabidopsis ZIP1 transporter (16, 19); therefore we
proceeded to determine with the hZIP1 probe if this transporter might
be expressed in the prostate cells. Fig.
5 demonstrates that both LNCaP and PC-3
cells exhibit expression of the putative hZIP1 transporter. Moreover,
the level of the putative ZIP1 mRNA was increased by treatment of
both cells with prolactin, indicative of hormonal regulation of a
hZIP1 transporter gene. A characteristic of some
ZIP transporter genes is the down-regulation by exposure of
cells to high zinc levels. Fig. 5 reveals that extended exposure of
PC-3 cells to zinc resulted in a decrease in the level of ZIP1 mRNA
indicative of a down-regulation of gene expression. The down-regulation by zinc adds to the evidence that we were probing the expression of a
zinc uptake transporter.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of prolactin and zinc on the
expression of hZIP1. Panel A, down-regulation of hZIP1
by zinc. PC-3 cells were grown in medium supplemented with
10 9 M prolactin to increase the expression of
hZIP1. The cells were then transferred to prolactin-free culture medium
containing 1 µg/ml zinc or no zinc (control) and incubated for
24 h. The cells were then collected, washed, and probed for hZIP1
mRNA. Panel B, prolactin effect on the level of hZIP1
mRNA. LNCaP and PC-3 cells were incubated for 1 h in medium
containing either 10 9 M prolactin or vehicle
(control). After incubation, the cells were quickly washed, prepared
for RNA extraction, and probed with 32P-labeled hZIP1
cDNA clone. 32P-Labeled cyclophilin cDNA probe and
the 28 S RNA band are presented as gel-loading controls.
|
|
 |
DISCUSSION |
The present studies demonstrate that the human malignant prostate
cells LNCaP and PC-3 possess the ability to accumulate high zinc
levels. The zinc levels of the prostate cells were significantly higher
than non-prostate squamous carcinoma cells. This parallels the studies
that demonstrated that rat prostate cells contained higher zinc levels
than non-prostate (kidney or liver) cells (7). In addition fibroblast
cell types reportedly contain about 0.4 fmol of zinc/cell (2).
Estimates of the cell content of zinc in LNCaP cells are minimally
(cells grown in "zinc-free" medium) about 1.0 fmol/cell, and in the
presence of medium supplemented with 1 µg/ml zinc, about 2.0 fmol/cell. When treated with prolactin or testosterone, the zinc
content is increased another 2-5-fold. Clearly, these cells accumulate
much higher zinc levels than non-prostate cells. Moreover, in other
unpublished studies we have demonstrated that when exposed to
extracellular zinc, the accumulation of zinc by PC-3 cells results in
the inhibition of their ability to oxidize citrate thereby causing in
citrate accumulation. This demonstrates that the accumulated zinc
includes a mobile reactive component that enters the mitochondria and
inhibits m-aconitase activity (8). To protect against the
toxic effects of zinc, mammalian cells generally employ defensive
mechanisms that prevent the accumulation of mobile reactive zinc (3).
In contrast, and consistent with their unique function, prostate
secretory epithelial cells employ mechanisms that facilitate the uptke
and accumulation of zinc.
The present studies demonstrate that prolactin and testosterone at
physiological concentrations regulate zinc uptake and accumulation in
LNCaP and PC-3 cells. Studies of zinc and citrate relationships with
rat prostate lobes revealed that prostate epithelial cells can be
characterized as the following types: V (ventral prostate), L (lateral
prostate), and D (dorsal prostate) (3, 7). An important distinguishing
characteristic is the response of the cells to prolactin and
testosterone in the regulation of zinc accumulation. Zinc accumulation
is decreased by both hormones in ventral prostate cells, increased by
both hormones in lateral prostate cells, and unaffected by either
hormone in dorsal prostate and non-prostate cells. Consequently, it
became important to establish which cell type(s) exists in human
prostate. The present studies reveal that both LNCaP and PC-3 cells
exhibit the characteristics of lateral prostate cells. This is
significant because rat lateral prostate is homologous to the lateral
lobes of the peripheral zone of the human prostate. This region of the
prostate is the dominant region for the origin of prostate malignancy
from which the LNCaP and PC-3 cells were originally derived; therefore
a linkage does exist between these cell lines and rat lateral prostate cells. However a most important relationship is that naturally occurring malignant prostate cells have lost the ability to accumulate zinc, whereas the malignant cell lines exhibit the capability of
accumulating zinc. It will be essential to establish the reason why
in situ malignant prostate cells do not accumulate zinc,
which will lead to an understanding of the pathogenesis of prostate cancer.
The present studies establish that a rapid zinc uptake transport
mechanism is associated with zinc accumulation by these cells and that
this transport mechanism is regulated by prolactin and by testosterone.
The initial kinetic studies indicate that the rapid transport would be
highly effective under the conditions reflective of circulating levels
of zinc. The most likely expectation is that prostate epithelial cells
possess a plasma membrane zinc transporter that permits the rapid
uptake of zinc from circulation. This would be similar to the unique
aspartate transporter that is involved in the accumulation of high
cellular levels of aspartate in prostate cells (21-23). We are now
proceeding with extensive kinetic studies to establish the
characteristics of this zinc transport process.
The fact that actinomycin completely abolished the stimulatory effects
of both hormones on zinc uptake by LNCaP and PC-3 cells and that
androgen receptor was required for testosterone stimulation of zinc
uptake provided initial evidence that gene regulation is involved.
Moreover, this is consistent with the hormonal regulation of the
aspartate transporter and other citrate-related metabolic genes in
prostate epithelial cells (4); therefore it seemed most plausible to
pursue the possibility that the kinetic demonstration of the rapid
uptake of zinc in the prostate cells might be the result of expression
of a plasma membrane-associated zinc uptake transporter. However,
little information is currently available regarding the expression of
zinc uptake transporters in mammalian cells. The expression and
operation of zinc transporters that export zinc out of cells and which
sequester intracellular zinc into organelles have been identified in
some mammalian cells (2, 24). In contrast, there has been no
established identification of any gene expression associated with
plasma membrane zinc uptake transporters in mammalian cells. Zinc
uptake transporters have been identified in plants, yeast, and other
organisms that comprise the homologous ZIP family of
transporter genes (16-19). Eide and colleagues (16, 19) preliminarily
identified two putative transporter genes in humans which are
homologous to the ZIP family of zinc uptake transporters, one of which
exhibits striking homology to Arabidopsis
ZIP1.2 The present studies
showed that the human homolog, hZIP1, is expressed in LNCaP and PC-3
cells and that the expression is hormonally regulated. This is
consistent with the hormonal regulation of the cellular accumulation of
zinc and the rapid zinc uptake in these cells. The present results also
demonstrate that the expression of hZIP1 is down-regulated
by exposure of cells to zinc, and this is characteristic of the zinc
regulation of ZIP1 and other zinc uptake transporters. The
Arabidopsis ZIP1 has been characterized as a plasma
membrane-associated zinc uptake transporter and as a rapid zinc uptake
transporter that leads to the cellular accumulation of zinc (19). Such
characteristics are surprisingly similar to the zinc uptake
characteristics of LNCaP and PC-3 cells identified in this study. The
striking similarities between the kinetic characteristics and hormonal
regulation of zinc uptake in the prostate cells and the ZIP transporter
provide compelling evidence for the expression and operation of a ZIP1
homolog (hZIP1) in prostate cells. This supports the contention (16,
19) that ZIP family zinc transporter genes are expressed in
human cells; however, we cannot yet represent conclusively that the
putative hZIP1 transporter is responsible for the rapid uptake
transport process that has been identified kinetically in the prostate
cells. It is conceivable that more than one zinc uptake transporter, as
is the case for yeast and plant cells (16-19), might be involved in
the uptake and accumulation of zinc in prostate epithelial cells. The
results of the present study now provide the basis for further studies
of the kinetic and genetic characterization of the zinc transport
mechanism(s) in normal and malignant prostate cells, the mechanism of
prolactin and testosterone regulation of zinc transport, the
relationship of zinc accumulation in the pathogenesis and progression
of prostate malignancy; they also provide a model system to study the
mechanisms of zinc transport in mammalian cells in general.
 |
ACKNOWLEDGEMENTS |
We express appreciation to Dr. David Eide
(Department of Biochemistry, University of Missouri) for valuable input
concerning zinc transporter genes and zinc transport mechanisms in
eukaryotic cells.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants CA 71207 and DK 28015.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: OCBS/Dental School,
University of Maryland, 666 W. Baltimore St., Baltimore, MD 21201. Tel.: 410-706-7618; Fax: 410-706-7618; E-mail:
lcc001{at}dental.umaryland.edu.
2
D. Eide, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
FBS, fetal bovine
serum;
HBSS, Hanks' balanced saline solution;
HUCL, Human Universal
cDNA Library;
PRL, prolactin.
 |
REFERENCES |
-
Reyes, J. G.
(1996)
Am. J. Physiol
270,
C401-C410[Abstract/Free Full Text]
-
Suhy, D. A.,
and O'Halloran, T. V.
(1995)
Metal Ions Biol. Syst.
32,
557-578
-
Valley, B. L.,
and Falchuk, K. H.
(1993)
Physiol. Rev.
73,
79-118[Free Full Text]
-
Franklin, R. B.,
and Costello, L. C.
(1997)
in
Prostate: Basic and Clinical Aspects (Naz, R. K., ed), pp. 115-150, CRC Press, Boca Raton, FL
-
Costello, L. C.,
and Franklin, R. B.
(1997)
Urology
50,
3-12[CrossRef][Medline]
[Order article via Infotrieve]
-
Costello, L. C.,
and Franklin, R. B.
(1998)
Prostate
35,
285-296[CrossRef][Medline]
[Order article via Infotrieve]
-
Liu, Y.,
Costello, L. C.,
and Franklin, R. B.
(1997)
Prostate
30,
26-32[CrossRef][Medline]
[Order article via Infotrieve]
-
Costello, L. C.,
Liu, Y.,
Franklin, R. B.,
and Kennedy, M. C.
(1997)
J. Biol. Chem.
272,
28875-28881[Abstract/Free Full Text]
-
Larue, J. P.,
and Morfin, R. F.
(1984)
Endocr. Res.
10,
171-181[Medline]
[Order article via Infotrieve]
-
Larue, J. P.,
and Morfin, R. F.
(1984)
Endocr. Res.
10,
183-192[Medline]
[Order article via Infotrieve]
-
Kavanagh, J. P.
(1983)
J. Reprod. Fertil
69,
359-363
-
Rosoff, B.
(1981)
in
The Prostatic Cell: Structure and Function (Murphy, G. P., Sandberg, A. A., and Karr, J. P., eds), pp. 447-457, Alan R. Liss, New York
-
Franklin, R. B.,
Juang, H. H.,
Zou, J.,
and Costello, L. C.
(1993)
Endocr. J.
3,
603-607
-
Juang, H. H.,
Costello, L. C.,
and Franklin, R. B.
(1995)
J. Biol. Chem.
270,
12629-12634[Abstract/Free Full Text]
-
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
-
Eide, D.
(1997)
Curr. Opin. Cell Biol.
9,
573-577[CrossRef][Medline]
[Order article via Infotrieve]
-
Zhao, H.,
and Eide, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2454-2458[Abstract/Free Full Text]
-
Zhao, H.,
and Eide, D.
(1996)
J. Biol. Chem.
271,
23203-23210[Abstract/Free Full Text]
-
Grotz, N.,
Fox, T.,
Connmoly, E.,
Park, W.,
Guerinot, M. L.,
and Eide, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7220-7224[Abstract/Free Full Text]
-
Franklin, R. B.,
Zou, J.,
Gorski, E.,
Yang, Y. H.,
and Costello, L. C.
(1997)
Mol. Cell. Endocrinol.
127,
19-25[CrossRef][Medline]
[Order article via Infotrieve]
-
Franklin, R. B.,
Lao, L.,
and Costello, L. C.
(1990)
Prostate
16,
137-146[Medline]
[Order article via Infotrieve]
-
Costello, L. C.,
Lao, L.,
and Franklin, R. B.
(1994)
Cell. Mol. Biol.
39,
515-524
-
Lao, L.,
Franklin, R. B.,
and Costello, L. C.
(1993)
Prostate
22,
53-63[Medline]
[Order article via Infotrieve]
-
Palmiter, R. D.,
and Findley, S. D.
(1995)
EMBO J
14,
639-649[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.