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 1 Department of Molecular Medicine, Northwest Hospital, Seattle, Washington 2 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. KEY WORDS: gap junctions; connexin43; prostate tumor; cell-cell communication INTRODUCTION Intercellular communication via gap junctions is believed to be involved in the regulation of cell growth . These membrane channels between contacting cells are composed of connexins (Cx), a highly conserved group of membrane proteins , that allow the passage of small molecules and ions up to 1,000 Daltons, some of which are presumed to act as growthregulatory signals . This function of gap junctions is highly evident in cancer development, where loss of communication competence is common among tumor cells . 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  are observed. In addition, factors required for tumorigenesis, including oncogenes and tumor promoters, inhibit GJC [8,9] while antineoplastic agents increase GJC . 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: email@example.com Received 11 March 1998; Accepted 5 May 1998 56 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 . Our data show a dramatic decrease in GJC as well as in expression and processing of Cx43 in neoplastic prostate cells. MATERIALS AND METHODS 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 . 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 microinjected. 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, MD). Immunofluorescence Detection of Cx43 For the immunodetection of Cx43 gap junctional plaques, cells were grown on glass coverslips in 8-well cell-culture trays . 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. RESULTS AND DISCUSSION 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 , 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 57 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 . 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 , 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 , 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. 3c). 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 ; and 3) impairment in the posttranslational processing of connexin . 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. 58 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.  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 . 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  and are abundantly found in communicating tissues and cells [15,16]. Constitutive expression of cell adhesion molecules  or inhibition of glycosylation  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 . Blockade of GJC in numerous neoplastic cells but not in carcinogen-initiated cells  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 . 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 , 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. ACKNOWLEDGMENTS The authors thank Dr. Sai L. Su (Northwest Biotherapeutics, Seattle, WA) for providing the TSU-Pr1 cell line, and M. Bates for photography. REFERENCES 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: 681–690. 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 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. lecke K, Yamasaki Y. Negative growth control of HeLa cells by connexin genes: Connexin species specificity. Cancer Res 1995; 55:629–639. 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; 10:1754–1763. 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– 64. Iizumi T, Yazaki T, Kanoh S, Kondo I, Koiso K. Establishment of a new prostatic carcinoma cell line (TSU-Pr1). J Urol 1987;137: 1304–1306. 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 1994;62:2394–2403. Hossain MZ, Ao P, Boynton AL. Rapid disruption of gap junctional communication and phosphorylation of connexin43 by 17. 18. 19. 20. 21. 22. 23. 25. 59 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– 529. 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.