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The Prostate 38:55–59 (1999)
Impaired Expression and Posttranslational
Processing of Connexin43 and Downregulation of
Gap Junctional Communication in Neoplastic
Human Prostate Cells
Mohammad Z. Hossain,1* Ajit B. Jagdale,1 Peng Ao,1 Cosette LeCiel,2
Ruo-Pan Huang,1 and Alton L. Boynton1
Department of Molecular Medicine, Northwest Hospital, Seattle, Washington
Pacific Northwest Cancer Foundation, Seattle, Washington
BACKGROUND. Gap junctional communication (GJC) has been implicated in the control of
cell proliferation. Numerous cancer cells show a decrease or loss of GJC compared to their
normal counterparts. Lack of adequate information on the status of gap junctions during
prostate neoplasia prompted us to examine this form of cancer, which comprises about 14%
of male cancer deaths in America.
METHODS. Cultured normal human prostate epithelial cells and several different human
prostate tumor lines were used in this study. GJC was assayed by dye transfer, whereas
Western blot and immunofluorescence methods were used to examine connexin43 (Cx43)
levels and the presence of gap junctions, respectively.
RESULTS. Normal human prostate cultures exhibited extensive cell-communication which
was completely absent in all the examined tumor cells. This disrupted communication was
associated with a decreased expression and an impaired posttranslational modification of
Cx43 in these cells. Abundant immunostaining of gap junctional channels by a Cx43-antibody
was observed in normal prostate cells but not in tumor cells.
CONCLUSIONS. Our data provide further support for the hypothesis that loss of junctional
communication is a critical step in progression to human prostate neoplasia. Prostate 38:55–59,
1999. © 1999 Wiley-Liss, Inc.
gap junctions; connexin43; prostate tumor; cell-cell communication
Intercellular communication via gap junctions is believed to be involved in the regulation of cell growth
[1]. These membrane channels between contacting
cells are composed of connexins (Cx), a highly conserved group of membrane proteins [2], that allow the
passage of small molecules and ions up to 1,000 Daltons, some of which are presumed to act as growthregulatory signals [1]. This function of gap junctions is
highly evident in cancer development, where loss of
communication competence is common among tumor
cells [3]. Conversely, when junctional communication
is reestablished in tumor cells following transfection
with connexins, a reversal of tumor phenotypes in
© 1999 Wiley-Liss, Inc.
vitro [4–6] as well as decreased tumor incidences in
vivo [7] are observed. In addition, factors required for
tumorigenesis, including oncogenes and tumor promoters, inhibit GJC [8,9] while antineoplastic agents
increase GJC [10]. Based on these observations, a tumor-suppressive role was suggested for gap junctional communication [2–4].
Grant sponsor: NIH/NCI; Grant number: CA57064.
Cosette LeCiel is currently at Zymogenetics, 1201 Eastlake Ave.
East, Seattle, WA 98102.
*Correspondence to: Mohammad Z. Hossain, Department of Molecular Medicine, Northwest Hospital, 120 Northgate Plaza, Suite
230, Seattle, WA 98125. E-mail:
Received 11 March 1998; Accepted 5 May 1998
Hossain et al.
To evaluate and extend our understanding of the
role of GJC in neoplasia, junctional communication in
cultured human normal and tumor prostate cells was
examined. Gap junctions in prostate cancer have not
been studied in detail, even though prostate cancer is
highly prevalent in men over age 50 and will claim
about 39,200 lives in the United States in 1998 [11]. Our
data show a dramatic decrease in GJC as well as in
expression and processing of Cx43 in neoplastic prostate cells.
Cell Culture
Primary human prostate epithelial cells were purchased from Clonetics (San Diego, CA) and were
grown as per the instructions of the supplier. Wellcharacterized neoplastic prostate cell lines, including
PC3, LNCaP, ALVA-31, and TSU-Pr1 [12–14], were
cultured in RPMI-1640 with 5% fetal calf serum.
Gap Junctional Communication Assay
Gap junctional communication was assessed by
transfer of the fluorescent dye Lucifer yellow after
single-cell microinjection, performed as described previously [10]. Briefly, glass micropipettes were prepared using a Flaming/Brown micropipette puller
(Sutter Instrument Co., Baltimore, MD) and backfilled
with a 10% solution of Lucifer yellow in 0.33 M LiCl.
Injection of cells in confluent cultures viewed by fluorescence illumination was achieved with pressure delivery by a microinjector. After 10 min, the number of
neighboring cells exhibiting dye labeling was recorded as an index of GJC. For each cell type, a minimum of 20–30 cells in different areas of cultures was
Western Blot Analysis of Cx43
Western blot analyses for Cx43 were performed as
described previously [15,16]. Briefly, confluent cells
were harvested in cold phosphate-buffered saline
(PBS) containing 1 mM Na-orthovanadate, 10 mM
NaF, and 1 mM phenylmethyl-sulfonyl fluoride
(PMSF). The cell pellets were lysed in 1 mM NaHCO3
containing 1 mM Na-orthovanadate, 10 mM NaF, and
1 mM PMSF. Cell lysates were resolved on polyacrylamide gels and transferred to polyvinyl difluoride
membranes. After blocking for 16 hr at 4°C in a solution containing 5% nonfat dry milk in Tris-buffered
saline and 0.2% Tween-20 (TBS-T), membranes were
incubated with an antibody recognizing the Cterminal region of Cx43. After several washes in TBS-
T, membranes were then incubated with horseradish
peroxidase-conjugated donkey anti-rabbit IgG and
processed with an enhanced chemiluminescence kit
(Kirkegaard and Perry Laboratories, Gaithersburg,
Immunofluorescence Detection of Cx43
For the immunodetection of Cx43 gap junctional
plaques, cells were grown on glass coverslips in 8-well
cell-culture trays [16]. Confluent cultures were rinsed
twice with PBS and then fixed in 3% formaldehyde for
20–30 min at room temperature. They were then permeabilized with 2% Triton X-100 and incubated in
freshly prepared 1% sodium borohydride for 15 min.
After washing with PBS, coverslips were incubated in
anti-Cx43 antibody for 2 hr and then washed with
PBS. After blocking for 30 min in normal goat serum,
cultures were incubated with fluorescein isothiocyanate FITC-conjugated goat anti-rabbit IgG antibody.
Coverslips were washed three times in PBS and
mounted onto glass slides with antifade medium, and
slides were visually evaluated using a Zeiss epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY)
and photographed.
Normal prostate epithelial cells showed high levels
of junctional communication (Fig. 1a–d). Immediately
after reaching confluence (5–6 days), they exhibited
moderate levels of dye transfer (Fig. 1a,b). Dye transfer was observed among 95% of the 30–40 injected
cells, and on average, 8–14 adjacent cells became dyepositive (mean, 12.4 ± 1.3). Several days later (day 10),
these cells exhibited variable compactness, with a
smaller cell size and a cuboid morphology (Fig. 1c). At
this stage, a dramatic rise in GJC was observed (Fig.
1d). The mean number of communicating cells increased to 85.5 ± 8.2. In contrast, dye transfer was
completely absent in neoplastic LNCaP cells (Fig. 1e,f).
We have examined several other established prostate
tumor lines, including PC3, ALVA-31, and TSU-Pr1
[12–14], and transfer of the microinjected fluorescent
dye could not be detected in any of these cell lines
(data not shown), except in TSU-Pr1, which showed
some degree of cell communication (mean, 4.7 ± 1.2).
Previous studies showed Cx43 as a constituent of
prostate gap junctions [17,18]. The alteration of Cx43
expression in prostate tumors is less well-understood.
Decreased Cx43 immunostaining was reported in
prostate tumors [16], while unchanged or increased
Cx43 levels were also observed [18,19]. To determine
whether the observed poor GJC in prostate tumor cells
is associated with alterations in Cx43 levels, we examined the expression of Cx43 in both normal and tumor
Impaired GJC in Neoplastic Prostate Cells
Fig. 2. Cx43 protein levels in normal and tumor prostate cells.
Equal amounts (10 µg) of cellular lysates were separated on SDSPAGE gels. The proteins were transferred to PVDF membrane,
and the membrane was probed with Cx43-antibody followed by
enhanced chemiluminescence detection. Different forms of Cx43
were labeled. Lane 1, WI38; lane 2, PC3; lane 3, LNCaP; lane
4, TSU-Pr1; lane 5, ALVA-31; lane 6, normal prostate epithelial
cells (6 days old); lanes 7 and 8, normal prostate epithelial cells
(10 days old). After ECL reaction, membranes were exposed for
1–2 min to visualize Cx43 expression in tumor cells. Lane 8 was
obtained after a short (15–20 sec) exposure of lane 7 to identify
different forms of Cx43 in normal prostate cells.
Fig. 1. Gap junctional communication in normal and tumor
prostate cells. Single cells in normal (a–d) or tumor (e,f) prostate
culture were microinjected with Lucifer yellow and 5 min later,
neighboring fluorescent cells were scored and photographed. a,b:
Six-day-old normal prostate epithelial cells. c,d: Ten-day-old normal prostate epithelial cells. e,f: Prostate tumor cell line LNCaP. a,
c, and e are phase-contrast photographs corresponding to the
fluorescent images b, d and f, respectively. Bar, 25 µm.
cells by Western blot analysis. In normal prostate cells,
Cx43 exists in three molecular forms (Fig. 2, lanes 6–8):
the 41-kD nonphosphorylated form (Cx43-NP), and a
43–45-kD doublet (Cx43-P1 and Cx43-P2), representing the phosphorylated forms of Cx43 observed in
other tissues and cell types [15,16]. The phosphorylation of Cx43 has been reported to be critical for the
establishment of functional gap junctions [20]. In moderately communicating 6-day-old cultures of nonneoplastic human prostate cells (Fig. 2, lane 6), two molecular forms of Cx43 (Cx43-NP and -P1) were predominant; in 10-day-old cultures (Fig. 2, lanes 7 and
8), we observed a several-fold increase in total Cx43
level as well as the presence of all three forms, which
correlated with the high GJC in these cells (Fig. 1c,d).
In another nonneoplastic human cell line, WI38 [21],
all three forms of Cx43 were also seen (Fig. 2, lane 1)
and correlated with its high cell communication abili-
ties (data not shown). In all the prostate tumor cells we
examined (Fig. 2, lanes 2–5), Cx43 expression was
drastically reduced, especially in PC3 and LNCaP
cells, where Cx43 expression was virtually undetectable. Another major difference between the nonneoplastic and the neoplastic prostate cells was the complete absence of any phosphorylated forms of Cx43 in
the latter.
Immunostaining of normal and tumor prostate cells
with anti-Cx43 antibody revealed a profile similar to
that of the Cx43 Western blots. In 6-day-old cultures of
nonneoplastic cells (Fig. 3a), Cx43 immunoreactivity
was mainly intracellular. In about 20% of cells, a punctate Cx43 staining was localized at cell-cell boundaries, indicating the presence of gap junctional
plaques [16], although in most of the cells, spotty Cx43
staining was present in a scattered fashion. In 10-dayold cultures of nonneoplastic cells, an overall increase
in Cx43 staining was observed (Fig. 3b). In these cells,
in addition to decreased intracellular staining, an increase in the size of Cx43-positive aggregates at cellcell boundaries was seen, which is in agreement with
the high communication efficiency of these cells. In
contrast, the prostate tumor cell line LNCaP was devoid of any positive staining at cell-cell boundaries
and only exhibited a faint intracellular staining (Fig.
Lack of junctional communication in tumor cells
has been linked with three types of connexin abnormality: 1) downregulation of connexin expression
[18,22]; 2) tyrosine-phosphorylation of connexin [8];
and 3) impairment in the posttranslational processing
of connexin [20]. Our results show a severe impairment in Cx43 expression and its posttranslational processing, resulting in a complete absence of gap junctions in neoplastic prostate cells, whereas normal
prostate epithelial cells are communication-efficient.
Hossain et al.
Fig. 3. Immunofluorescent staining of Cx43 in fixed cultures.
Cells grown on glass coverslips were fixed in 3% formaldehyde
and, following Triton permeabilization, were incubated with antiCx43 antibody. Binding of Cx43 antibody was visualized by FITCconjugated goat anti-rabbit IgG antibody. a: Six-day-old normal
prostate culture. b: Ten-day-old normal prostate culture. c: Prostate tumor cell line LNCaP. Arrowheads in a and b indicate positive staining of gap junctional plaques at the cell-cell boundary. Bar,
12 µm.
The loss of junctional competence in all examined
prostate tumor cell lines indicates that it is perhaps a
required step in prostate neoplasia. Although Mehta
et al. [19] observed Cx43 expression in neoplastic but
not in normal prostate cells, our findings are in accord
with two reports showing the presence of Cx43 in normal human prostate [17,18], which decreased in highgrade prostatic tumors [17]. Moreover, the communication abilities of both normal and tumor prostate cells
are in complete agreement with their corresponding
levels of Cx43 expression observed in this study. The
mechanism of the downregulation of connexin expression is presently unknown. Transcriptional regulation
of eukaryotic genes is generally modulated by proteinDNA interaction at specific nucleotide sequences at
the promoter region; characterization of Cx response
elements as well as identification of tumor-specific
regulatory proteins will be required for solving this
puzzle. Another reason for the communication deficiency in tumor cells appears to be obstructed posttranslational steps of Cx43 that generate Cx43phosphoforms which are necessary for the formation
of functional gap junctions [20] and are abundantly
found in communicating tissues and cells [15,16]. Constitutive expression of cell adhesion molecules [20] or
inhibition of glycosylation [23] has been shown to
overcome this obstruction. It will be interesting to examine whether similar approaches are useful in correcting abnormal Cx43 processing in neoplastic prostate cells, which are usually devoid of E-cadherin expression [24].
Blockade of GJC in numerous neoplastic cells but
not in carcinogen-initiated cells [10] indicates a critical
role of GJC in the development of the neoplastic phenotype. It has been postulated that by shutting down
the communication channels, a tumor cell can completely isolate itself from the control of growthregulatory signals produced by surrounding normal
cells and can thus undergo aberrant cell proliferation
to eventually yield a tumor [25]. Although the identity
of such growth-regulatory molecules traversing gap
junctions is yet to be determined, reversion of tumorigenic phenotypes following restoration of junctional
communication [4–6] supports this hypothesis. Since
the reversion of the neoplastic phenotype has been
suggested to be dependent upon connexin and tissue
types [6], it remains to be seen whether the neoplastic
phenotype of prostate cells can be reverted by an increased expression and/or normalization of posttranslational processing of Cx43.
The authors thank Dr. Sai L. Su (Northwest Biotherapeutics, Seattle, WA) for providing the TSU-Pr1
cell line, and M. Bates for photography.
1. Loewenstein WR, Rose B. The cell-cell channel in the control of
growth. Semin Cell Biol 1992;3:59–79.
2. Bruzzone R, White TW, Paul DL. Connection with connexins:
The molecular basis of direct intercellular signaling. Eur J Biochem 1996;238:1–27.
3. Yamasaki H, Naus CCG. Role of connexin genes in growth control. Carcinogenesis 1996;17:1199–1213.
4. Chen SC, Pelletier DB, Ao P, Boynton AL. Connexin43 reverses
the phenotype of transformed cells and alters their expression of
cyclin/cyclin-dependent kinases. Cell Growth Differ 1995;6:
5. Zhu D, Caveney S, Kidder GM, Naus CCG. Transfection of C6
glioma cells with connexin43 cDNA: Analysis of expression,
intercellular coupling, and cell proliferation. Proc Natl Acad Sci
USA 1991;88:1883–1887.
6. Mesnil M, Krutovskikh V, Piccoli C, Elfgang C, Traub O, Wil-
Impaired GJC in Neoplastic Prostate Cells
lecke K, Yamasaki Y. Negative growth control of HeLa cells by
connexin genes: Connexin species specificity. Cancer Res 1995;
Naus CCG, Elisevitch K, Zhu D, Belliveau DJ, Del Maestro RF.
In vivo growth of C6 glioma cells transfected with connexin43
cDNA. Cancer Res 1992;52:4208–4213.
Crow DS, Beyer EC, Paul DL, Kobe SS, Lau AF. Phosphorylation
of Cx43 gap junction protein in uninfected and Rous sarcoma
virus-transformed mammalian fibroblasts. Mol Cell Biol 1990;
Oh SY, Grupen GS, Murray AW. Phorbol ester induces phosphorylation and down-regulation of connexin43 in WB cells.
Biochim Biophys Acta 1991;1094:243–248.
Hossain MZ, Wilkens LR, Mehta PP, Loewenstein WR, Bertram
JS. Enhancement of gap junctional communication by retinoids
correlates with their ability to inhibit neoplastic transformation.
Carcinogenesis 1989;10:1743–1748.
Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics,
1998. CA 1998;48:6–29.
Loop SM, Rozanski TA, Ostenson RC. Human prostate tumor
line, ALVA-31: A new model for studying the hormonal regulation of prostate tumor cell growth. Prostate 1993;22:93–108.
Webber MM, Bello D, Quader S. Immortalized and tumorigenic
adult human prostatic epithelial cell lines: Characteristics and
applications. Part 2. Tumorigenic cell lines. Prostate 1997;30:58–
Iizumi T, Yazaki T, Kanoh S, Kondo I, Koiso K. Establishment of
a new prostatic carcinoma cell line (TSU-Pr1). J Urol 1987;137:
Hossain MZ, Murphy LJ, Hertzberg EL, Nagy JI. Phosphorylated forms of connexin43 predominate in rat brain: Demonstration by rapid inactivation of brain metabolism. J Neurochem
Hossain MZ, Ao P, Boynton AL. Rapid disruption of gap junctional communication and phosphorylation of connexin43 by
platelet-derived growth factor in T51B rat liver epithelial cells
expressing PDGF receptor. J Cell Physiol 1998;174:66–77.
Tsai H, Werber J, Davia MO, Edelman M, Tanaka KE, Melman
A, Christ GJ, Gelieber J. Reduced connexin43 expression in high
grade, human prostatic adenocarcinoma cells. Biochem Biophys
Res Commun 1996;227:64–69.
Wilgenbus KK, Kirkpatrick CJ, Knuechel R, Willecke K, Traub
O. Expression of Cx26, Cx32 and Cx43 gap junction proteins in
normal and neoplastic human tissues. Int J Cancer 1992;51:522–
Mehta PP, Lokeshwar BL, Schiller PC, Bendix MV, Ostenson
RC, Howard GA, Roos BA. Gap junctional communication in
normal and neoplastic prostate epithelial cells and its regulation
by cAMP. Mol Carcinog 1996;15:18–32.
Musil LS, Cunningham BA, Edelman GM, Goodenough DA.
Differential phosphorylation of the gap junction protein Cx43 in
junctional communication-competent and -deficient cell lines. J
Cell Biol 1990;111:2077–2088.
Tupper JT, Smith JW. Growth factor regulation of membrane
transport in human fibroblasts and its relationship to stimulation of DNA synthesis. J Cell Physiol 1985;125:443–448.
Lee SW, Tomasetto C, Paul D, Keyomarsi K, Sager R. Transcriptional downregulation of gap junctional proteins blocks junctional communication in human mammary tumor cell lines. J
Cell Biol 1992;118:1213–1221.
Wang Y, Mehta PP, Rose B. Inhibition of glycosylation induces
open connexin43 cell-cell channels and phosphorylation and
Triton X-100 insolubility of connexin43. J Biol Chem 1995;270:
26581–26585.24. Umbas R, Schalken JA, Aalders TW, Carter BS,
Karthaus HFM, Schaafsma HE, Debruyne FMJ, Isaaca WB. Expression of cell adhesion molecule is reduced or absent in highgrade prostate cancer. Cancer Res 1992;52:5104–5109.
Mehta PP, Bertram JS, Loewenstein WR. Growth inhibition of
transformed cells correlates with their junctional communication with normal cells. Cell 1986;44:187–196.
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