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• W2053171430 abstract "Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions. In primary culture, connexin-43 is expressed by thyrocytes in monolayers or reorganized into follicles, whereas the expression of connexin-32 is dependent upon the reconstitution of follicles. To study the functional impact of connexin-32 gap junctions in thyroid cells, we transfected connexin-32 cDNA in two thyroid-derived communication-deficient cell lines, FRT and FRTL-5. The selected clones, which stably expressed connexin-32 at high levels and exhibited high gap junction-mediated dye-coupling, presented a reduced proliferation rate as compared with that of the corresponding wild-type FRT and FRTL-5 cells; the mean population doubling time was increased by ∼35%. The proliferation of connexin-32-transfected FRTL-5 cells remained thyrotropin-dependent; the range of thyrotropin concentrations that stimulated growth was the same in transfected and control cells. The expression of connexin-32 led to an increase of thyroglobulin gene expression in FRTL-5 cells. The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells. Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions. In primary culture, connexin-43 is expressed by thyrocytes in monolayers or reorganized into follicles, whereas the expression of connexin-32 is dependent upon the reconstitution of follicles. To study the functional impact of connexin-32 gap junctions in thyroid cells, we transfected connexin-32 cDNA in two thyroid-derived communication-deficient cell lines, FRT and FRTL-5. The selected clones, which stably expressed connexin-32 at high levels and exhibited high gap junction-mediated dye-coupling, presented a reduced proliferation rate as compared with that of the corresponding wild-type FRT and FRTL-5 cells; the mean population doubling time was increased by ∼35%. The proliferation of connexin-32-transfected FRTL-5 cells remained thyrotropin-dependent; the range of thyrotropin concentrations that stimulated growth was the same in transfected and control cells. The expression of connexin-32 led to an increase of thyroglobulin gene expression in FRTL-5 cells. The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells. Gap junctions (GJ) are ubiquitous intercellular junctions allowing the cell-to-cell exchange of small cytoplasmic molecules. These exchanges have long been thought to play a role in the regulation of cell growth and cell differentiation in a number of tissues (1Loewenstein W.R. Biochim. Biophys. Acta. 1979; 560: 1-65Crossref PubMed Scopus (891) Google Scholar, 2Guthrie S.C. Gilula N.B. Trends Neurosci. 1989; 12: 12-16Abstract Full Text PDF PubMed Scopus (196) Google Scholar). GJ proteins, connexins (Cx), 1The abbreviations used are: GJ, gap junction(s); Cx, connexin(s); TSH, thyrotropin; kbp, kilobase pair(s); TTF-1, thyroid transcription factor-1.belong to a multigenic family, each member presenting its own tissue-specific distribution. Despite strong overall homologies between Cx, functional properties (including permeability properties) differ from one Cx to the other. In most tissues, the determination of the role of a given Cx is complicated by the coexpression of several other different Cx (for reviews, see Refs. 3Dermietzel R. Hwang T.K. Spray D.S. Anat. Embryol. 1990; 182: 517-528Crossref PubMed Scopus (128) Google Scholar, 4Bennett M.V.L. Barrio L.C. Bargiello T.A. Spray D.C. Hertzberg E. Saez J.C. Neuron. 1991; 6: 305-320Abstract Full Text PDF PubMed Scopus (860) Google Scholar, 5Beyer E.C. Int. Rev. Cytol. 1993; 137C: 1-37PubMed Google Scholar, 6Kumar N.M. Gilula N.B. Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar, 7Bruzzone R. White T.W. Paul D.L. Eur. J. Biochem. 1996; 238: 1-27Crossref PubMed Scopus (1244) Google Scholar). This applies to endocrine glands (8Stagg R.B. Fletcher W.H. Endocr. Rev. 1990; 11: 302-325Crossref PubMed Scopus (136) Google Scholar, 9Munari-Silem Y. Rousset B. Eur. J. Endocrinol. 1996; 135: 251-264Crossref PubMed Scopus (61) Google Scholar) including the thyroid. Previous work from our laboratory has established that polarized thyroid epithelial cells or thyrocytes present an unusual Cx expression pattern; they coexpress Cx32 and Cx43. Cx32 GJ are scattered along the lateral domain of the plasma membrane, and Cx43 GJ are precisely located within the tight junction network (10Guerrier A. Fonlupt P. Morand I. Rabilloud R. Audebet C. Krutovskikh V. Gros D. Rousset B. Munari-Silem Y. J. Cell Sci. 1995; 108: 2609-2617PubMed Google Scholar). Using pig thyrocytes in primary culture, which, under defined culture conditions, either spread in monolayers or reconstitute follicles, we have shown that the Cx43 gene is always expressed. By contrast, the Cx32 gene is expressed only in thyrocytes that reorganize into follicles (11Munari-Silem Y. Guerrier A. Fromaget C. Rabilloud R. Gros D. Rousset B. Endocrinology. 1994; 135: 724-734Crossref PubMed Scopus (42) Google Scholar). Cx32 GJ and Cx43 GJ that reconstitute in thesein vitro systems are functional, and thyrotropin (TSH) controls both Cx synthesis and the level of junctional coupling between thyrocytes (11Munari-Silem Y. Guerrier A. Fromaget C. Rabilloud R. Gros D. Rousset B. Endocrinology. 1994; 135: 724-734Crossref PubMed Scopus (42) Google Scholar, 12Munari-Silem Y. Audebet C. Rousset B. Endocrinology. 1991; 128: 3299-3309Crossref PubMed Scopus (46) Google Scholar). The respective roles of Cx32 GJ and Cx43 GJ in thyroid gland functioning are not known. Cx32 GJ could play a prominent role since Cx32 expression correlates with the expression of thyroid histiotypic differentiation, i.e. follicle formation. To try to document the functional impact of cell-to-cell communication via Cx32 GJ, we have chosen to transfect two rat thyroid-derived cell lines (FRT and FRTL-5) that are communication-deficient with the rat Cx32 cDNA. Several clones that stably expressed high levels of Cx32 have been isolated from each cell line. All of them presented a high level of GJ-mediated cell-to-cell communication and exhibited a decreased growth rate as compared with the corresponding parental cells. Interestingly, stable expression of Cx32 by differentiated FRTL-5 cells led to up-regulation of the thyroglobulin gene. The 1.5-kb pCx32 cDNA (containing the entire coding region) was isolated from the pGEM-3 plasmid (kindly provided by Dr. D. L. Paul) (13Paul D.L. J. Cell Biol. 1986; 103: 123-134Crossref PubMed Scopus (522) Google Scholar) by EcoRI site digestion. Cx32 cDNA was inserted into the pSVK3 plasmid (Pharmacia Biotech Inc.) after linearization and dephosphorylation at its uniqueEcoRI site, yielding pSVK3-Cx32. The proper orientation of the cDNA insert was controlled by electrophoretic analysis ofKpnI and PstI restriction enzyme digestion fragments and nucleotide sequencing of the appropriate region. FRT and FRTL-5 cells were kindly provided by Prof. L. Nitsch (Dipartimento di Biologia e Patologia Cellulare e Moleculare “L. Califano,” Universita Degli Studi di Napoli Federico II, (Naples, Italy). These two cell lines, derived from normal thyroids of Fischer rats, have been previously characterized (14Ambesi-Impiombato F.S. Parks L.A.M. Coon H.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3455-3459Crossref PubMed Scopus (976) Google Scholar, 15Ambesi-Impiombato F.S. Picone R. Tramontano D. Cold Spring Harbor Conf. Cell Proliferation. 1982; 9: 483-492Google Scholar). FRTL-5 cells were grown in complete culture medium composed of Coon's modified Ham's F-12 medium (Seromed, Berlin, Germany) containing 100 units/ml penicillin and 100 μg/ml streptomycin and supplemented with 5% calf serum and a five-hormone mixture (10 μg/ml insulin, 10 nmhydrocortisone, 5 μg/ml transferrin, 10 ng/ml glycyl-l-histidyl-l-lysine acetate, and 1 milliunits/ml TSH) as described (15Ambesi-Impiombato F.S. Picone R. Tramontano D. Cold Spring Harbor Conf. Cell Proliferation. 1982; 9: 483-492Google Scholar). All hormones were from Sigma. FRT cells were cultured in the same medium as FRTL-5 cells, except that TSH was omitted and calf serum was replaced by fetal calf serum (Sigma). Cell cultures were maintained at 37 °C under a 95% air and 5% CO2 humidified atmosphere and were routinely subcultured by trypsinization with a change of medium twice weekly. FRT and FRTL-5 cells (∼3–5 × 105 cells/100-mm Petri dish) were cotransfected with two plasmids (pSVK3-Cx32 and pCMV-neo) using the calcium phosphate precipitation procedure followed by a glycerol shock. The optimal conditions of transfection were slightly different for the two cell lines. FRT cells were incubated for 3–4 h with calcium phosphate in the presence of 20–30 μg of pSVK3-Cx32 and 2–3 μg of pCMV-neo and then subjected to a 1-min glycerol shock. For FRTL-5 cells, the incubation in the presence of calcium phosphate was reduced to 1 h, and then cells were subjected to a 3-min glycerol shock. For both cell types, 48 h after the outset of transfection, the neomycin analogue, G418 (Life Technologies, Inc.), was added to the medium at a concentration of 0.4 mg/ml. The medium containing G418 was changed every 3 days. After 3 weeks of selective pressure in the presence of G418, neomycin-resistant FRT colonies were picked by trypsinization in cloning cylinders and grown separately under selective conditions. In the case of FRTL-5 cells, only neomycin-resistant colonies judged to be well coupled by microinjection of lucifer yellow (see “Measurement of Intercellular Communication”) were picked up and grown for subsequent analyses. For controls, cells were cotransfected under the conditions described above, but with pCMV-neo and the pSVK3 vector lacking Cx32 cDNA. FRT and FRTL-5 transfectants were maintained in the appropriate media containing G418 at a concentration of 0.05 mg/ml. cDNAs encoding rat Cx26 (clone Cx26-1; 1.1 kbp), rat Cx32 (1.5 kbp), and rat Cx43 (clone G1; 2.5 kbp) originate from Drs. B. J. Nicholson (16Zhang J.-T. Nicholson B.J. J. Cell Biol. 1989; 109: 3391-3401Crossref PubMed Scopus (362) Google Scholar), D. L. Paul (13Paul D.L. J. Cell Biol. 1986; 103: 123-134Crossref PubMed Scopus (522) Google Scholar), and E. C. Beyer (17Beyer E.C. Paul D.L. Goodenough D.A. J. Cell Biol. 1987; 105: 2621-2629Crossref PubMed Scopus (921) Google Scholar), respectively. cDNA clone G1 contains 92% of the coding region for Cx43; the other two cDNAs contain the complete coding regions of Cx26 and Cx32. G1 Cx43 cDNA was extracted from the Bluescript plasmid using EcoRI and then digested with StuI (Promega); thisEcoRI-StuI fragment (1.03 kbp; positions 293–1323) was used for hybridization experiments. Cx32 cDNA as well as Cx26 cDNA were extracted from the pGEM-3 plasmid byEcoRI site digestion. The thyroglobulin cDNA probe corresponds to the human thyroglobulin 0.7-kbp M1 fragment (18Malthiery Y. Lissitzky S. Eur. J. Biochem. 1987; 165: 491-498Crossref PubMed Scopus (277) Google Scholar). The rat Pax-8 cDNA fragment (0.3 kbp), corresponding to the paired domain fragment (19Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (275) Google Scholar), was extracted from the C27 B2xx22 plasmid byEcoRI and HindIII restriction site digestion. The rat TTF-1 cDNA fragment (0.6 kbp), corresponding to the 3′-untranslated region, was extracted from the Bluescript THA plasmid (20Guazzi S. Price M. De Felice M. Damante G. Mattei M.G. Di Lauro R. EMBO J. 1990; 9: 3631-3639Crossref PubMed Scopus (472) Google Scholar). cDNA probes, prepared from DNA fragments isolated by electrophoresis and purified using the QIAEX gel extraction kit (QIAGEN Inc.), were labeled with [α-32P]dCTP by random hexanucleotide primed synthesis. Total RNA was isolated from wild-type FRT and FRTL-5 cells, stable transfected clones, and control rat tissues (liver, cervix, and heart) using the guanidinium isothiocyanate/acid phenol extraction method of Chomczynski and Sacchi (21Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63290) Google Scholar). Northern blot analysis of total RNA (20 μg/sample) was performed as described (22Fromaget C. El Aoumari A. Dupont E. Briand J.P. Gros D. J. Mol. Cell. Cardiol. 1990; 22: 1245-1258Abstract Full Text PDF PubMed Scopus (50) Google Scholar). Hybridized membranes were exposed to Kodak X-Omat AR film (Eastman Kodak Co.) at −80 °C with intensifying screens. The efficiency of transfer and the integrity of RNA were checked by hybridization with a β-actin probe. Cells attached to Petri dishes fixed with 4% paraformaldehyde in phosphate-buffered saline containing 0.25% Triton X-100 for 30 min at 20 °C were incubated with anti-Cx32 antibodies diluted 1:1000 in phosphate-buffered saline containing 1 mg/ml bovine serum albumin for 1 h at 20 °C. Anti-Cx32 antibodies were generated in rabbit against a synthetic peptide corresponding to the sequence of the cytoplasmic loop of rat Cx32 (residues 98–124) (10Guerrier A. Fonlupt P. Morand I. Rabilloud R. Audebet C. Krutovskikh V. Gros D. Rousset B. Munari-Silem Y. J. Cell Sci. 1995; 108: 2609-2617PubMed Google Scholar). A goat anti-rabbit Ig F(ab′)2fragment conjugated to fluorescein (Sigma) was used as secondary antibody. Immunofluorescent images were taken using a SIT camera (LHESA Electronique, Cercy Pontoise, France) installed on an Axiophot microscope (Zeiss, Oberkochen, Germany) coupled to an image-processing system (Sapphire, Quantel, Montigny-le-Bretonneux, France). Photomicrographs were prepared using a video printer (UP 5000 P, Sony, Tokyo). Cells at confluence in 100-mm culture dishes were collected in cold Earle's medium (pH 7.0) by scraping. After centrifugation, the cells were resuspended in 1 mmNaHCO3 supplemented with 1 mmphenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 10 mm NaF, 2 μg/ml leupeptin, and 1 μg/ml pepstatin (buffer A) and lyzed by sonication. Cell lysates were centrifuged at 100,000 × g for 30 min at 4 °C, and the resulting pellets were resuspended in buffer A containing 0.25%N-lauroylsarcosine sodium salt (Sigma) and incubated for 20 min at 20 °C as described previously (10Guerrier A. Fonlupt P. Morand I. Rabilloud R. Audebet C. Krutovskikh V. Gros D. Rousset B. Munari-Silem Y. J. Cell Sci. 1995; 108: 2609-2617PubMed Google Scholar). After centrifugation at 100,000 × g for 30 min at 4 °C, the final pellets were resuspended in 50 mm Tris, 0.01% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, 2% (w/v) SDS, and 10 mg/ml bromphenol blue (pH 6.7). Proteins were separated by electrophoresis on SDS-12% acrylamide gel and transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA). The transfer membrane was saturated in Blotto solution (40 mm Tris, 5% (w/v) skim milk, and 0.1% (v/v) Tween 20) for 1 h at room temperature and then incubated with rabbit anti-Cx32 antibodies (1:500 final dilution in Blotto) for 2 h at room temperature. After extensive washings in 10 mm Tris, 150 mm NaCl, and 0.2% Tween 20 (pH 8.0), immunocomplexes were visualized using alkaline phosphatase-conjugated anti-rabbit IgG and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrates (Sigma) according to the manufacturer's instructions. Western blot analyses of thyroglobulin were performed on total cell extracts. Cell-to-cell communication was analyzed using the low M rfluorescent probe lucifer yellow CH (Sigma), originally described by Stewart (23Stewart W.W. Cell. 1978; 14: 741-759Abstract Full Text PDF PubMed Scopus (968) Google Scholar). The probe was microinjected into one cell, and after diffusion to adjacent cells, the number of lucifer yellow-labeled cells was counted as described previously (12Munari-Silem Y. Audebet C. Rousset B. Endocrinology. 1991; 128: 3299-3309Crossref PubMed Scopus (46) Google Scholar). Cells were seeded at low density in multiwell culture dishes. After different culture times, cells were dissociated by mild trypsinization, and the number of cells/dish was counted under an inverted microscope equipped with phase-contrast optics using an hemocytometer. FRT and FRTL-5 cells do not express the Cx expressed in normal thyrocytes. No Cx32 transcript was detected in total RNA from FRT and FRTL-5 cells by Northern blotting (Fig.1 A, lanes 2and7, respectively); likewise, Cx43 and Cx26 transcripts could not be found (data not shown). The same negative results were also obtained at the protein level. Neither Cx32 (Fig. 1 B,lanes 2and 7) nor Cx43 (data not shown) was detected by Western blot analyses of FRT or FRTL-5 detergent-resistant membrane extracts. Immunofluorescence labeling with anti-Cx32 (Fig.2, A and E) or anti-Cx43 (data not shown) antibodies was also negative. The inability to detect any of the three Cx (Cx32, Cx43, and Cx26) was in keeping with the absence of or the very low level of junctional coupling analyzed by lucifer yellow microinjection. In FRTL-5 cells, lucifer yellow was never transmitted from the injected cells to surrounding cells as illustrated Fig. 3 B(panel b). In FRT cells, the dye was sometimes detected in some cells (two to five cells) adjacent to the injected cell several minutes after microinjection (Fig. 3 A, panel b). The low level of coupling observed in FRT cells could be either related to the presence of a low number of GJ channels composed of Cx32, Cx43, or Cx26 expressed in minute amounts or dependent on channels composed of another Cx than those normally expressed in thyroid cells.Figure 2Immunofluorescence detection of Cx32 in transfected FRT and FRTL-5 cells. Indirect immunofluorescence labeling was performed on fixed and permeabilized cells attached to Petri dishes using anti-peptide antibodies as described under “Experimental Procedures.” A, wild-type FRT cells;B, Cx32-FRT cells (clone A); C, Cx32-FRT cells (clone B); D, Cx32-FRT cells (clone C); E, wild-type FRTL-5 cells; F, Cx32-FRTL-5 cells (clone I);G, Cx32-FRTL-5 cells (clone P); H, Cx32-FRTL-5 cells (clone Q). Bars = 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Evidence that intercellular communication is restored in Cx32-transfected FRT and FRTL-5 cells. Lucifer yellow was injected into the cytoplasm of one cell (identified by thearrows), and the distribution of the fluorescent probe was examined 5 min after microinjection. Panels a andc correspond to phase-contrast images, and panels b and d give the fluorescence images of the corresponding fields. A, FRT cells. Panels aandb, wild-type cells; panels c and d, Cx32-transfected cells (clone A). B, FRTL-5 cells.Panels aand b, wild-type cells; panels cand d, Cx32-transfected cells (clone Q).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cx32-transfected FRT cells were identified by the presence of the 1.6-kb Cx32 mRNA. Among ∼70 clones, the three clones expressing the highest levels of Cx32 mRNA (clones A, B, and C) (Fig. 1 A) were selected and used for subsequent studies. The Cx32 mRNA content of clones A and C (Fig. 1 A,lanes 4and 6) was higher than that of clone B (lane 5). FRTL-5 cells expressing the Cx32 gene were identified by their high junctional coupling. Three clones (clones I, P, and Q) were selected; they expressed similar levels of Cx32 transcripts (Fig. 1 A, lanes 9–11). Cx32 mRNA was absent in FRT and FRTL-5 cells cotransfected with the neomycin gene and the empty pSVK3 vector (Fig. 1 A, lanes 3and8). The expression of the Cx32 gene in FRT and FRTL-5 cells was further analyzed by Western blotting and immunofluorescence labeling. The Cx32 protein of the expected size (27–28 kDa) was immunodetected in all the Cx32 mRNA-positive clones (Fig.1 B, lanes 4–6 for FRT cell-derived clones andlanes 9–11 for FRTL-5 cell-derived clones). As expected from mRNA data, the amount of Cx32 protein in clone B of FRT cells was lower than that detected in both clones A and C (Fig.1 B, compare lane 5 with lanes 4and6). In transfected FRTL-5 cells, the amount of Cx32 protein was similar in the three clones (Fig. 1 B, lanes 9–11). Fig. 2 illustrates the cellular distribution of Cx32 in transfected FRT and FRTL-5 cells. The immunofluorescence labeling of Cx32-transfected FRT cells was qualitatively similar for the three clones (Fig. 2,B–D); it appeared as discontinuous lines and/or dots delineating the region of cell-cell contacts. Bright dots were also visible over the cells. In Cx32-transfected FRTL-5 cells, the labeling profile was different. In the three clones (Fig. 2, F–H), the labeling appeared as rather large and round spots more randomly distributed over the cells. As FRTL-5 cells did not spread very well on the Petri dish, but rather remained tightly grouped, the location of the labeled spots was more difficult to establish; fluorescent spots were found in the regions of cell-cell contacts, but also in other regions of the plasma membrane or possibly inside the cells. In both cell types, Cx32-transfected cells were intensely labeled. The level of Cx32 expression (assessed by mRNA and protein measurements) remained stable beyond 30 and 20 passages in transfected FRT and FRTL-5 cells, respectively. These results show that the Cx32- transfected cells we selected stably overexpressed the exogenous rat Cx32 gene, the major part of the protein being addressed to the plasma membrane. Cx32-transfected cells exhibited a high level of GJ-mediated intercellular communication. When microinjected into one cell, lucifer yellow was detected within seconds in numerous cells in the vicinity of the microinjected one; this is illustrated for FRT cell-derived and FRTL-5 cell-derived clones in Fig. 3 (A andB, respectively). The level of cell-to-cell communication or junctional coupling of each clone was quantified; the results are presented in the form of histograms of frequency (Fig.4) that allow the collection and comparison of data from multiple microinjection tests on the different clones. Among the Cx32-transfected FRT cells, clones A and C, which presented the highest level of Cx32 expression, showed the highest dye-coupling capacity. Indeed, >50% of the cells from clones A and B and only 15% from clone C communicated with >20 neighbor cells. The three clones of Cx32-transfected FRTL-5 cells exhibited similar junctional coupling levels. These data unequivocally establish that Cx32 protein synthesized by FRT and FRTL-5 cells from the exogenous Cx32 cDNA is competent to form functional gap junction channels. In this study, FRT and FRTL-5 cells that stably express Cx32 are designated Cx32-FRT and Cx32-FRTL-5, respectively. The proliferation rate of Cx32-transfected cells was compared with that of control cells (wild-type cells and cells expressing the neomycin resistance gene: FRT-neo or FRTL-5-neo). The results of Fig.5 A show that the proliferation rate of the three clones of Cx32-FRT cells was significantly lower than that of control cells. Interestingly, cells from clone B (the clone among the three that expressed the lowest level of Cx32 and intercellular communication) had an intermediate growth rate between that of control cells and that of cells from the two other clones (clones A and C). The growth rate of the three clones of Cx32-FRTL-5 cells was also markedly reduced as compared with that of control cells. After a 10-day culture period, the population of Cx32-FRTL-5 cells represented less than half of that of wild-type FRTL-5 cells. TableI gives the average population doubling time calculated for each clone in different experiments. One can observe that the mean doubling time was increased by ∼35% in two of the three clones of Cx32-FRT cells and in the three clones of Cx32-FRTL-5 cells (as compared with that of the corresponding parental wild-type cells).Table IPopulation doubling time of FRT and FRTL-5 cells transfected with the rat Cx32 geneClonesPopulation doubling timehFRT cells Wild-type23.2 ± 1.1 Neo23.7 ± 1.6 Cx32-A31.4 ± 1.61-aStatistically different from controls (p < 0.05). Cx32-B25.4 ± 1.8 Cx32-C31.5 ± 2.71-aStatistically different from controls (p < 0.05).FRTL-5 cells Wild-type36.9 ± 3.0 Neo36.4 ± 0.4 Cx32-I50.8 ± 3.61-aStatistically different from controls (p < 0.05). Cx32-P51.2 ± 3.31-aStatistically different from controls (p < 0.05). Cx32-Q45.9 ± 0.51-aStatistically different from controls (p < 0.05).Wild-type FRT cells, FRT cells transfected with the neomycin gene only (clone neo) or cotransfected with the neomycin and Cx32 genes (clones Cx32-A, Cx32-B, and Cx32-C), wild-type FRTL-5 cells, and FRTL-5 cells transfected with the neomycin gene (clone neo) or cotransfected with the neomycin and Cx32 genes (clones Cx32-I, Cx32-P, and Cx32-Q) were cultured in duplicate wells for 2, 4, 6, 8, and 10 days. At each time point, cells were dissociated by mild trypsinization and counted. The population doubling time (PDT) was calculated according to the following formula: PDTt = t·ln 2/ln(Nt/N i), whereN i is the plating cell number andNt is the cell number at time t. The population doubling time of a given clone in a given experiment was the mean of the PDTt values measured at days 2, 4, 6, 8, and 10. Results represent the mean ± S.E. of three to four separate experiments.1-a Statistically different from controls (p < 0.05). Open table in a new tab Wild-type FRT cells, FRT cells transfected with the neomycin gene only (clone neo) or cotransfected with the neomycin and Cx32 genes (clones Cx32-A, Cx32-B, and Cx32-C), wild-type FRTL-5 cells, and FRTL-5 cells transfected with the neomycin gene (clone neo) or cotransfected with the neomycin and Cx32 genes (clones Cx32-I, Cx32-P, and Cx32-Q) were cultured in duplicate wells for 2, 4, 6, 8, and 10 days. At each time point, cells were dissociated by mild trypsinization and counted. The population doubling time (PDT) was calculated according to the following formula: PDTt = t·ln 2/ln(Nt/N i), whereN i is the plating cell number andNt is the cell number at time t. The population doubling time of a given clone in a given experiment was the mean of the PDTt values measured at days 2, 4, 6, 8, and 10. Results represent the mean ± S.E. of three to four separate experiments. FRTL-5 cells are characterized by their TSH-dependent growth (15Ambesi-Impiombato F.S. Picone R. Tramontano D. Cold Spring Harbor Conf. Cell Proliferation. 1982; 9: 483-492Google Scholar). As illustrated in Fig.6 A, the proliferation of FRTL-5 cells is arrested upon TSH withdrawal. When TSH was omitted from the culture medium, the growth of Cx32-FRTL-5 cells was also totally blocked, indicating that the proliferation of the communication-competent cells remained TSH-dependent. To determine whether the reduction in the rate of proliferation of Cx32-FRTL-5 cells was related to a change in their TSH responsiveness, we compared the growth rate of Cx32-transfected and wild-type FRTL-5 cells in response to increasing concentrations of TSH. The concentration-response curves shown in Fig. 6 B indicate that the concentration of TSH required to obtain 50% of the maximum growth rate was very similar for transfected and nontransfected cells (∼50 microunits/ml). Proliferation of FRTL-5 cells is known to be activated by insulin. In this study, insulin (10 μg/ml) was systematically added to the FRTL-5 culture medium. The difference in growth between transfected and nontransfected cells was similar in the presence and absence of insulin (Fig. 6 C). By varying the serum concentration from 0.5 to 5%, the difference in proliferation between wild-type and Cx32-FRTL-5 cells was maintained (Fig. 6 D). FRTL-5 cells expressed several thyroid-specific genes, including the genes encoding thyroglobulin, the thyroid prohormone, and two transcription factors (TTF-1 and Pax-8), as shown in Fig.7 (lane 1in A–C, respectively). TTF-1 and Pax-8 mRNA levels were similar in Cx32-FRTL-5, wild-type FRTL-5, and control FRTL-5-neo cells; but a huge increase in the amount of thyroglobulin mRNA was reproducibly observed in the three clones of Cx32-FRTL-5 cells (Fig. 7 A, compare lanes 3–5 with lanes 1and2). The increase of thyroglobulin gene expression in Cx32-transfected cells was confirmed at the protein level. The Western blot analysis results reported in Fig. 7 E show that thyroglobulin was present in higher amounts in Cx32-FRTL-5 cells (lanes 3–5) than in control cells (lanes 1and2). FRT cells expressed Pax-8, but neither TTF-1 nor thyroglobulin. The level of Pax-8 mRNA was similar in Cx32-FRT and control cells (data not shown). This study clearly demonstrates that, as a consequence of transfection with and expression of the exogenous Cx32 gene that led to the restoration of cell-to-cell communication, the rate of proliferation of FRT and FRTL-5 cells was significantly reduced. In all the selected clones, the levels of Cx32 mRNA and Cx32 protein as well as the level of junctional coupling, on one hand, and the reduction of growth, on the other, remained stable beyond 20 or 30 passages (in Cx32-FRT and Cx32-FRTL-5 cells, respectively), indicating a stable insertion of the cDNA into the genome of host cells. Insertion of th" @default.
• W2053171430 created "2016-06-24" @default.
• W2053171430 creator A5003923518 @default.
• W2053171430 creator A5006333741 @default.
• W2053171430 creator A5007038370 @default.
• W2053171430 creator A5027410914 @default.
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• W2053171430 date "1997-09-01" @default.
• W2053171430 modified "2023-09-26" @default.
• W2053171430 title "Restoration of Cell-to-Cell Communication in Thyroid Cell Lines by Transfection with and Stable Expression of the Connexin-32 Gene" @default.
• W2053171430 cites W106000010 @default.
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