Reduced levels of connexin43 in cervical dysplasia: inducible expression in a cervical carcinoma cell line decreases neoplastic potential with implications for tumor progression

Timothy J. King1,2,5, Laurie H. Fukushima1, A. David Hieber3, Kelly A. Shimabukuro1, Wael A. Sakr4 and John S. Bertram1,6

1 Molecular Carcinogenesis, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI 96813,
2 Cell, Molecular and Neurosciences Program, Department of Cell and Molecular Biology, University of Hawaii at Manoa, Honolulu, HI 96822,
3 Department of Plant Molecular Physiology, University of Hawaii at Manoa, Honolulu, HI 96822 and
4 Pathology Department, Harper Hospital, Wayne State University, Detroit, MI 48201, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Loss of gap junctional intercellular communication (GJIC) has been linked to aberrant proliferation and an enhanced neoplastic phenotype. Many human tumors, including the cervical carcinoma line HeLa, have been reported to be deficient in expression of the gap junction protein connexin43 (Cx43) and GJIC. To determine if this is an early event in carcinogenesis, we utilized immunohistochemistry to screen a series of cervical biopsy samples and demonstrated a major reduction in Cx43 expression in dysplastic regions compared to normal epithelia. To determine whether this loss influences the neoplastic behavior of cervical carcinoma cells, we have constructed HeLa cell lines in which Cx43 expression can be induced in response to doxycycline. This approach allows for the discrimination of Cx43-mediated effects from those due to pre-existing clonal heterogeneity. Cx43 induction in these cells led to assembly of functional junctions but did not alter growth control in vitro as measured by logarithmic growth, saturation density or focus formation when in co-culture with growth-controlled fibroblasts. However, Cx43 induction decreased two indices of neoplasia: it reduced anchorage-independent growth and attenuated the growth rate of tumor xenografts. These results indicate that established HeLa cell lines are unresponsive to Cx43-mediated signals which are thought to mediate growth control of non-transformed cells, however, Cx43 expression can still reduce aspects of the neoplastic phenotype of these cells, indicating that loss of connexin signaling in dysplastic cells may contribute to their neoplastic progression.

Abbreviations: ATRA, all-trans retinoic acid; BME, basal Eagle's medium; BSA, bovine serum albumin; Cx43, connexin43; DiO, 3,3'-dioctadecyloxacarbocyanine perchlorate; Dox, doxycycline HCl; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GJIC, gap junctional intercellular communication; NHF, normal human fibroblasts; PBS, phosphate-buffered saline without calcium; PMSF, phenylmethylsulfonyl fluoride; rtTA, tetracycline-responsive transactivator.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gap junctional intercellular communication (GJIC) allows for direct transmission between neighboring cells of ions and small hydrophilic molecules <1–2 kDa in size; these include metabolites and messengers such as sodium, potassium, calcium, cAMP, ATP and inositol 1,4,5-triphosphate (13). Transmission occurs by passive diffusion through aqueous channels which span the plasma membranes of two adjacent cells. These channels are formed by the docking of two hemi-channels (connexons), each contributed by one of the adjoining cells; hemi-channels are comprised of a hexameric arrangement of proteins termed connexins which form the channel (35). Connexins are a group of at least 14 highly conserved proteins which demonstrate development- and tissue-specific expression patterns (3,4,6). This family of proteins plays an important role in normal development and physiology and a loss of function is implicated in various human syndromes, including inherited cardiac abnormalities (7), non-syndromic deafness (8), cataracts (9) and peripheral nerve disorders (10). Several studies involving transgenic mouse strains with germline-inactivated connexin genes (knockouts) have revealed the importance of connexins in normal developmental and physiological processes: connexin43 (Cx43) knockouts display lethal cardiac malformation (11); connexin32 knockouts have compromised livers which exhibit a higher rate of spontaneous and chemical carcinogenesis (12); connexin26 knockouts die in utero because of an apparent failure of placental transfer of nutrients to developing embryos (13); female connexin37 knockout mice fail to ovulate most probably due to a lack of GJIC-mediated signals between granulosa cells and immature oocytes (14).

Just as the presence of connexins and presumably GJIC has been implicated in the control of normal development and growth, the absence of GJIC in human and animal cells has been associated in multiple studies with decreased growth control and tumorigenesis (reviewed in refs 3,4,6). In several studies lack of expression or function was demonstrated in human tumors, namely prostate (15,16), liver (17), ovary (18) and astrocytoma (19), although the situation is unclear in the breast where increased (20) and decreased (21) expression of connexins has been reported. To our knowledge, expression has not been evaluated in pre-neoplastic cells. It is unclear how much of this loss of connexin expression is a consequence of selective pressures during tumor growth or passage in cell culture. If loss of expression and/or function is a frequent early event in tumors in situ, this would add weight to the hypothesis that loss of GJIC contributes to carcinogenesis. This question is of particular relevance to the mechanism of action of two classes of potential cancer preventive agents, the retinoids and carotenoids, which have been shown to increase Cx43 expression and GJIC in mouse and human fibroblasts and in human keratinocytes in culture (2224), while retinoic acid has been shown to increase Cx43 expression in human skin (25). This action has been statistically correlated with the chemopreventive activities of these agents in experimental models of carcinogenesis and it has been proposed that the enhanced transfer of growth-inhibitory signals as a consequence of the action of these agents is antiproliferative and contributes to their chemopreventive capacity (2628). Moreover, induction of GJIC between growth-inhibited normal and transformed cells results in growth inhibition of the latter (29). To address the question of whether loss of connexin expression is an early event in carcinogenesis, we have examined routine diagnostic cervical biopsy samples obtained after cytology tests which indicated the presence of squamous cervical dysplasia, considered a precursor of cervical carcinoma. This site was chosen because of the high frequency of such lesions in the population, the predicted expression of Cx43 in this epithelium consistent with its expression in human skin (25) and because retinoic acid has been shown to act as a chemopreventive agent against low grade cervical lesions (30), while carotenoids have been associated with decreased risk of cancer at this site (31,32). As an additional rationale, the human cervical carcinoma cell line HeLa has been reported to be deficient in Cx43 expression yet capable of assembling functional gap junctions after restoration of exogenous Cx43 expression following gene transfer (33,34).

We report here that while Cx43 is indeed expressed in suprabasal layers of the normal cervical epithelium, this expression is strongly reduced in regions of dysplasia, areas which also exhibit aberrant proliferation and irregular morphology. To address the possible consequences of this loss of expression, HeLa cells have been engineered to re-express Cx43 under the control of an inducible promoter. When induced, these cells express functional gap junctions and this induction results in decreases in the neoplastic potential of these cells. This decrease was manifest as diminished ability to grow in an anchorage-independent environment in vitro and reduced growth rates of subcutaneous tumors injected into an immunodeficient mouse xenograft model. To our knowledge this is the first successful use of an inducible connexin gene expression system which can be regulated under both in vitro and in vivo situations. The use of inducible cell lines avoids interpretational problems associated with clonal heterogeneity that we and others have encountered in studies utilizing constitutive promoters, although the possibility of non-specific effects resulting from overexpression of any exogenous gene product still remains. The major advantage of this approach is that when uninduced each cell line can act as its own control allowing for unambiguous evaluation of the effects from expression of a single exogenous gene. Until recently, the lack of suitable regulatable promoters has impeded such investigations, however, the development by Gossen and Bujard of the tetracycline-responsive mammalian gene expression system, which has been extensively used in HeLa cells, suggested a way to overcome this problem of clonal heterogeneity (35,36). Here we show that loss of Cx43 expression, and presumably of GJIC, is an early event in cervical carcinogenesis and that re-expression of this gene in cervical carcinoma cells is associated with decreased neoplastic potential. These data strongly support the notion that connexins can function as tumor suppressor genes and also support the hypothesis of growth control by junctionally communicated regulatory signals first proposed by Loewenstein (37,38), with the caveat that long-established tumor cell lines may, through selective pressure, lose the ability to generate or respond to such signals. The two major unanswered questions are: the nature of the connexin-mediated signals and the molecular mechanism by which Cx43 is down-regulated in pre-neoplastic and neoplastic cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Immunohistochemical detection of Cx43 in cervical biopsies
A series of 10 archival formaldehyde fixed and paraffin embedded biopsy samples, submitted sequentially to Harbor Hospital Pathology Laboratory for pathological analysis of dysplasia, were stained for Cx43 protein levels using a rabbit polyclonal antibody (F26) directed against the C-terminal 19 amino acids of the predicted human Cx43 sequence. This antibody had been shown to detect a 43 kDa protein in western blotting experiments with protein lysate obtained from human fibroblasts (Figure 1F) and keratinocytes (not shown) previously demonstrated to express Cx43. Immunoreactivity was shown to be abolished by prior incubation with the immunizing peptide. In addition, immunohistochemical studies of Cx43-expressing tissues have shown immunoreactivity to junctional plaques consistent with the location of gap junctions (Figure 1CGo). Biopsy samples were stripped of patient identifiers and were determined exempt from informed consent requirements. Because of the sequential acquisition of these biopsy samples, there was no patient bias for age or ethnicity. Samples were deparaffinized, hydrated without the requirement for antigen retrieval protocols, incubated with the primary anti-Cx43 antibody at a 1:100 dilution and subsequently stained with a commercial ABC kit (Vector Laboratories). Nuclei were subsequently counterstained with methylene blue. All preparations were performed on the same day using the same reagents.



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Fig. 1. Immunohistochemical detection of Cx43 in archival cervical biopsy samples. Red staining is indicative of Cx43 immunoreactivity. (A) Normal epithelium. Note the absence of Cx43 expression in the single layer of basal cells and the progressively increasing expression in apical cells undergoing terminal differentiation. (B) Cervical dysplasia (CIN 2). Cx43 expression is excluded from approximately the lower five layers of cells and even in the apical cells, which are immunopositive, staining is significantly less than that seen in the normal epithelium shown in (A). Note the high nuclear:cytoplasmic ratio in these Cx43 non-expressing cells and the presence of inflammatory cells in the underlying stroma. A magnified view of this dysplastic epithelium is shown in (C). Note the two mitotic figures in the suprabasal cells, staining of the perimeter of cells consistent with the expected location of connexons and the virtual absence of immunostaining in the lower six layers of cells. (D) A section of normal epithelium in which the primary antibody was pre-immune serum; note the lack of immunostaining in the suprabasal layers. (E) A fragment of dysplastic epithelium exhibiting little or no Cx43 expression in the lower five to six cell layers. The stage of CIN is difficult to judge in this section because of possible loss of the apical layers. (F) Western blot of proteins extracted from human fibroblasts with a molecular weight range of ~200–14 kDa detected with the same anti-Cx43 antibody at a 1:1000 dilution. The upper band in the 43 kDa region typically represents phosphorylated isoforms of Cx43 (22); note the lack of immunoreactivity in other regions of the blot.

 
Cell lines and culture techniques
All cell lines were cultured in basal Eagle's medium (BME) (Gibco BRL) containing 25 µg/ml gentamicin supplemented with 2–5% fetal bovine serum (certified tetracycline-free; Clontech) or 2–5% bovine calf serum (Hyclone) and incubated at 37°C in a 5% CO2/air atmosphere. Cell dissociation was performed using trypsin:EDTA (Gibco BRL). The HeLa-On reverse mutant tetracycline-responsive transactivator (rtTA)-expressing cell line was purchased from Clontech. Primary human fibroblast cell strains used in co-culture experiments were isolated by trypsinization from mechanically dispersed neonatal foreskins with subsequent passaging equivalent to other cell lines. C3H10T1/2 immortalized embryonic mouse fibroblasts (39) were utilized for co-culture experiments and as a positive control in northern analysis of Cx43 (not shown).

Transfection and cloning
The cDNA coding for rat cardiac Cx43 was excised from plasmid pG2A (40) by EcoRI restriction digestion and subcloned bi-directionally into the EcoRI site of the rtTA-responsive plasmid pUHD10-3 (obtained from S. Reed, La Jolla) (35,41,42) creating the Cx43 sense orientation construct pUHDCx43(sense). Inducible expression of the Cx43 cDNA from the pUHDCx43(sense) construct is under the control of a minimal CMV (cytomegalovirus) immediate-early promoter (PmCMV) downstream from a septad of tet operator sequences (35,36,42).

The HeLa-On line is the result of stable transfection of the human cervical carcinoma HeLa cell line with a construct containing both a gene encoding rtTA, constructed from the reverse mutant tetracycline receptor fused to the herpes simplex virus VP-16 transcription transactivator domain (36), as well as a gene encoding G418 resistance (Clontech). Inducible-Cx43 HeLa clones were obtained by co-transfecting variable amounts of pUHDCx43(sense) (5–60 µg/100 mm culture dish) along with plasmid p3ss' (lacR deleted; Stratagene) (0.1 µg/100 mm dish), encoding hygromycin resistance, into the HeLa-On cell line employing calcium phosphate precipitation techniques (Profection; Promega). Cells were subsequently selected for 3 weeks in medium containing 200 µg/ml hygromycin (Sigma) and ring cloned into separate dishes. Clones were periodically maintained in medium containing 350 µg/ml G418 (Gibco BRL) or 200 µg/ml hygromycin. Cells were initially screened by SDS–PAGE and immunoblot analysis following 48 h induction with 1 µg/ml of the tetracycline analog doxycycline HCl (Dox) (Sigma) (36).

SDS–PAGE and western immunoblot of induced Cx43
Uninduced and induced (1 µg/ml Dox, 48 h) cultures were harvested in phosphate-buffered saline without calcium (PBS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM NaF, pelleted and disrupted in lysis buffer (borate buffer, pH 8.0) containing 1% Nonidet P-40 and 1 mM PMSF at 2.5x107 cells/ml for 1 h at 4°C. Cell debris was pelleted and the protein supernatant was quantified using the BCA Assay (Pierce Chemical Co.). Equal concentrations of protein lysates were reduced for 1 h in 1x SDS–PAGE sample buffer containing 10% ß-mercaptoethanol and 10% SDS, loaded in equal amounts per lane and run on 10% SDS–polyacrylamide gels (22). Proteins were electrophoretically transferred to Immobilon PVDF membrane (Millipore Co.) and immunodetected using the Western-Light Chemiluminescent Detection Kit (Tropix Inc.). The membrane was blocked for 1 h and subsequently incubated for 1 h at room temperature with a rabbit polyclonal antibody (1:500 dilution) directed against the C-terminus of Cx43 (residues 368–382) (22), followed by detection and autoradiography according to the manufacturer's directions. Equal protein loading between lanes was confirmed by Coomassie blue staining of membranes following detection. Quantification of autoradiograph band densities was performed using computer image densitometry (NIH Image). Identical techniques were used with the F26 antibody (1:1000 dilution) to detect Cx43 in human fibroblasts (Figure 1F).

Northern blotting
Cell line RNA analysis. Total RNA was isolated using the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX) from 107 cells uninduced or induced for 48 h with 1 µg/ml Dox. Samples (20 µg) were denatured in formaldehyde (24) and separated on 0.9% agarose gels before being transferred to a nylon membrane (Schleicher & Scheull). Nylon filters were hybridized using a 1400 bp cDNA probe to rat Cx43 (40). Double-stranded cDNA probes were radiolabeled with [32P]dCTP (3000 Ci/mmol) using a commercial random primer labeling kit (Gibco BRL). Filters were prehybridized at 4°C for 3 h in 50% formamide, 5x SSPE (1x SSPE = 150 mM NaCl, 10 mM NaH2PO4, pH 7.4), 5x Denhardts, 1% SDS and 100 µg/ml denatured salmon sperm DNA and then hybridized in the same buffer containing 10% dextran sulfate and the cDNA probe (5x106 c.p.m./ml) at 42°C overnight. Filters probed for Cx43 transcripts were then washed twice in 1x SSPE, 0.1% SDS at room temperature, once in 0.2x SSPE, 0.1% SDS at 42°C for 20 min and once in 0.2x SSPE, 0.1% SDS at 45°C for 20 min and then exposed to X-ray film. Equal loading of RNA was confirmed by digital analysis of ethidium bromide stained rRNA (18S).

Tumor RNA analysis. Excised tumors were flash frozen in liquid nitrogen and stored at –70°C for subsequent analysis of tumor RNA. Tumor samples (0.1 mg) were homogenized and RNA was extracted, electrophoresed and detected as described above for cell line RNA analysis. Tumor RNA blots were stripped (50% formamide, 1% SDS for 60 min at 70°C) and subsequently reprobed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (43) expression levels as described above. Blots were digitally analyzed to normalize total RNA loading between lanes using GAPDH transcript levels.

Detection of assembled Cx43-containing gap junctions by indirect immunofluorescence microscopy
Cell culture. Cells were seeded at 2.5x105 cells/well on plastic slides (2 cm2/well, Permanox; Nunc Inc.) for 8 h and subsequently induced by addition of medium with or without 1 µg/ml Dox for 48 h. For analysis of colonies growing in soft agar, colonies were removed with a pipette and spread onto a slide. Slides were then rinsed in PBS and fixed in methanol overnight at –20°C. Slides were rinsed in acetone at –20°C, blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at 4°C, then incubated with the anti-Cx43 rabbit polyclonal antibody described previously for western analysis of HeLa lines (1:30 dilution in a 5% BSA solution) in a humidity chamber for 1 h at 4°C. Slides were then washed in PBS at room temperature for 1 h with an additional PBS wash for 10 min followed by a 1 h incubation with a fluorescein isothiocyanate-labeled anti-rabbit secondary antibody (1:80 dilution in a 5% BSA solution; Sigma). Finally, slides were rinsed three times in PBS followed by a rinse in 0.1 M Tris–HCl (pH 8.0) and then mounted in 90% glycerol (pH 8.0) containing 3.4 mg/ml 1,4-diazabicyclo-[2,2,2]octane (Sigma) to prevent photobleaching. Photomicrographs were obtained on a Zeiss Axioplan microscope.

Tumor sections. Tumors were fixed in formalin, embedded in paraffin and sectioned into 5 µm slices by standard procedures with subsequent deparaffinization and immunofluorescent detection as described above.

Detection of homologous GJIC by scrape-loading
Communication was measured as transfer of the fluorescent dye Lucifer yellow CH (dilithium salt; Sigma) from scrape-loaded cells to cells distal to the incision (44). High density cultures, uninduced and induced with 1 µg/ml Dox for 48 h, were rinsed with PBS, covered with 0.2% Lucifer yellow in PBS and an incision was made through the monolayer with a sterile scalpel. Cultures were incubated for 5 min to allow dye uptake and subsequently rinsed with PBS. The medium was replaced for 8 min under incubation conditions to allow for dye transfer between communicating cells followed by a final PBS rinse and photography. Results of duplicate experiments are reported.

Detection of GJIC by microinjection
To verify the presence of GJIC among induced HeLa cells and between induced HeLa cells and fibroblasts in co-culture experiments, HeLa-On, HIC100 and HIC122 cells, uninduced and induced with 2 µg/ml Dox for 24 h, were pre-labeled for identification purposes with a membrane-specific, fluorescent lipophilic dye, 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) (10 mM stock in dimethylformamide, absorbance 484 nm, emission 501 nm) at a concentration of 100 µM in 0.3 M glucose for 1 h. Cells were rinsed, trypsinized, replated overnight in medium to ensure survival and subsequently seeded (5x104 cells/60 mm dish) onto confluent C3H10T1/2 mouse fibroblast monolayers pre-incubated in medium with or without 2 µg/ml Dox. Labeled HeLa cells were then microinjected with the fluorescent dye Lucifer yellow CH (10% in 0.33 M LiCl) as described (45) and communication was measured as transfer of the dye from DiO-labeled HeLa cells to communicating DiO-labeled HeLa cells (homologous GJIC) or adjacent communicating fibroblasts (heterologous GJIC). Approximately 1 and 10 min after microinjection, dye transfer to adjacent cells was recorded by photography. Results of duplicate experiments are reported.

Logarithmic and saturation density growth assays
Logarithmic growth assay. HIC122 cells, uninduced or induced for 24 h with 1 µg/ml Dox, were seeded (2x104 cells/60 mm dish) in duplicate in the presence or absence of 1 µg/ml Dox and counted every 24 h post-seeding.

Saturation density growth assay. HeLa-On and HIC122 cells were seeded (5x105 cells/60 mm dish) in duplicate in the presence or absence of 1 µg/ml Dox for 60 h and then counted every 12 h. Cells were quantified by Coulter counter and reported as the means of duplicate dishes. Qualitatively similar results were obtained in triplicate experiments.

Co-culture growth assay (inducible-Cx43 HeLa cells/growth-controlled fibroblasts)
HeLa-On, HIC89, HIC100 and HIC122 cells, untreated or treated with 1 µg/ml Dox for 48 h, were seeded in triplicate (200 cells/60 mm dish) onto confluent monolayers of C3H10T1/2 fibroblasts, primary normal human fibroblasts (NHF) or empty control dishes in BME containing 3.5% calf serum with or without 1 µg/ml Dox. After 7 days, cells were rinsed with PBS, fixed and stained with Giemsa (Sigma). Foci containing ~100 cells or more were scored positive. These experiments were repeated three times with qualitatively similar results.

Anchorage-independent growth assay (soft agar)
Two thousand cells/clone (HeLa-On, HIC100,122), untreated or treated with 2 µg/ml Dox for 48 h, were seeded in duplicate in 0.4% agarose (Type II; Sigma) medium (BME containing 5% fetal bovine serum; Clontech) with or without 2 µg/ml Dox onto a solid 0.8% agarose/medium layer (with or without Dox) previously solidified on the bottom of each 60 mm dish. Cultures were maintained by weekly medium additions with or without 2 µg/ml Dox for a total of 4 weeks. Colonies were visualized by the addition of 1.5 mg/ml NitroBlue tetrazolium vital dye (Sigma) for 24 h. The minimal colony size scored was ~5 µm3. This experiment was repeated three times with qualitatively equivalent results.

Tumorigenicity in immunodeficient mice
Male nude mice (Crl:CD-1-nuBR) were purchased from Charles River Laboratories. The experimental protocol was approved by the University of Hawaii's Institutional Animal Care and Use Committee. Immunodeficient mice, untreated (5% sucrose water) or pre-treated with sucrose water containing Dox (0.2 mg/ml Dox, 5% sucrose) 2 days prior to injection, were s.c. inoculated with 2.5x106 cells/flank in 0.1 ml of BME medium containing 3.5% calf serum (Hyclone). Induction of Cx43 expression was maintained by feeding animals water containing Dox, protected from light degradation by dark feeder bottles, which was replenished every 3–4 days. Tumor volume was measured every 3 days with a caliper in two perpendicular dimensions. After 16–25 days, tumors were excised immediately following sacrifice, divided in two and either fixed in cold buffered 10% formalin or flash frozen in liquid nitrogen and stored at –70°C for subsequent analysis of tumor RNA as described above. Tumor volume was calculated assuming a spherical shape: volume = 4/3({pi})r3.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cx43 protein level is decreased in cervical dysplasia
Immunohistochemical analysis of 10 consecutive cervical biopsy samples submitted for pathological diagnosis to the Pathology Department of Harper Hospital, Wayne State University, was conducted utilizing a polyclonal antibody directed against the 19 C-terminal residues of the deduced sequence of human Cx43. In pathologically normal epithelium, Cx43 was seen to be strongly expressed in suprabasal regions but was excluded from the single layer of germinal cells (Figure 1AGo). This restricted expression is identical to that reported for human skin (25). In regions showing dysplasia, little or no expression was detected in approximately the lower five layers of epithelium (Figure 1BGo), in contrast to the single layer in normal cervix, and in those outer cell layers demonstrating expression the intensity of immunostaining was greatly reduced in comparison with normal tissue. A high power view of such an area is shown in Figure 1CGo to demonstrate that staining is restricted to regions of cell–cell apposition. Because of the clinical sampling procedures utilized, several specimens consisted of entirely dysplastic regions with no clear evidence of orientation. Only low levels of Cx43 expression were detectable in these lesions except in small areas containing cells exhibiting normal morphology. In one case, the biopsy contained tissue fragments showing normal morphology (not shown) and a fragment with clear evidence of dysplasia (Figure 1EGo): Cx43 expression was decreased in the dysplastic sample, but not in that with normal morphology, indicating that loss of expression is a local phenomenon associated with premalignant changes. No staining was observed when pre-immune serum was used as primary antibody (Figure 1DGo). The reduction in Cx43 immunostaining in regions of dysplasia was consistently observed in all cases of dysplasia examined and thus appears to be a frequent event in pre-malignant lesions of the cervix. In further support of the specificity of this antibody, Figure 1F shows a western blot of proteins extracted from cultured human fibroblasts demonstrating immunoreactivity only in the appropriate 43 kDa region.

Creation of inducible-Cx43 HeLa cell lines and western immunoblot evaluation
To determine the influence of re-expression of Cx43 in cervical carcinoma cells, we created a series of genetically engineered cell lines in which exogenous Cx43 can be induced by exposure to Dox using the tetracycline-responsive inducible gene expression system designed by Gossen and Bujard (35,36). The commercially available HeLa-On cell line was stably co-transfected with the pUHDCx43(sense) construct containing rat Cx43 cDNA and a second plasmid encoding a hygromycin resistance selection marker. A total of 40 hygromycin/G418-resistant clones were analyzed by western immunoblot for Cx43 expression following induction with 1 µg/ml Dox for 48 h. Figure 2Go shows the Cx43 protein levels of five clones, four of which exhibit inducible Cx43 (HIC89, 100, 114 and 122) and one (HIC101) which, representing the majority of clones screened, was non-responsive to Dox treatment. As can be seen, high levels of Cx43 induction can be achieved in these four clones with only a minimal basal leak when uninduced. Densitometry of autoradiographs revealed relative Cx43 induction levels on the order of 15- to 35-fold over uninduced levels (for example HIC89 = 16-fold, HIC122 = 35-fold). Multiple isoforms of Cx43, which presumably differ in the extent of phosphorylation (46), are clearly evident for these four clones, suggesting post-translational modification of the exogenous Cx43 protein within these cell lines. Although the parental HeLa-On cell line was shown to express very low levels of endogenous Cx43 (lane 1), these levels did not change in response to Dox treatment (data not shown) nor was the HeLa-On line junctionally competent (data not shown).



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Fig. 2. Engineered HeLa lines exhibit Dox-inducible Cx43 protein expression: western blot analysis. The HeLa-On parental line and five G418/hygromycin-resistant clones uninduced or induced with 1 µg/ml Dox for 48 h are shown. Immunoblots (40 µg protein/lane) were detected with a polyclonal antibody directed against rat Cx43. –, uninduced; +, induced. Lane 1, HeLa-On parental line; lanes 2 and 3, HIC89; lanes 4 and 5, HIC100; lanes 6 and 7, HIC114; lanes 8 and 9, HIC122; lanes 10 and 11, HIC101.

 
Northern analysis of inducible-Cx43 HeLa lines
These inducible lines were evaluated for induction of Cx43 mRNA by northern blotting of total RNA. As shown in Figure 3Go, 48 h induction with Dox results in high levels of Cx43 mRNA production in the three inducible clones analyzed (HIC100, 114 and 122) with a prominent band at ~2.2 kb, as would be expected for mRNA transcripts produced from the exogenous rat Cx43 cDNA (40). Uninduced cultures exhibited minor levels of exogenous Cx43 mRNA (2.2 kb) representing a small leak from PmCMV in the absence of Dox. A similar induction profile was seen for HIC89 (data not shown). Detection of a low level of endogenous Cx43 transcript (3.1 kb) in the HeLa-On and inducible cell lines is consistent with the low Cx43 protein level observed by western analysis in the absence of Dox (Figure 2Go, lane 1). In addition, no change in endogenous Cx43 transcript levels (3.1 kb) was observed following Dox treatment of the parental HeLa-On line (lane 5) nor was any 2.2 kb transcript detected in the treated or untreated situations (lanes 1 and 5). RNA from C3H10T1/2 cells used as a positive control exhibited a 3.1 kb transcript size consistent with previous reports (22), illustrating probe specificity (data not shown). Ethidium bromide staining of the gel confirmed equal loading of RNA between lanes (data not shown).



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Fig. 3. Engineered HeLa lines display Dox-inducible Cx43 mRNA expression: northern analysis. Total RNA was purified from clones uninduced or induced with 1 µg/ml Dox for 48 h and electrophoresed, transblotted and detected with a radiolabeled cDNA probe to rat Cx43. Lanes 1–4, uninduced (–); lanes 5–8, induced (+). Lanes 1 and 5, HeLa-On parental line; lanes 2 and 6, HIC100; lanes 3 and 7, HIC114; lanes 4 and 8, HIC122. Arrows denote endogenous 3.1 kb and exogenous 2.2 kb Cx43 transcripts.

 
Maximal protein induction was achieved by 0.5–1.0 µg/ml Dox within 24–36 h (data not shown). Cellular toxicity, observed as an increased number of floating cells, was not seen until levels of between 8 and 16 µg/ml Dox were reached. Dox-induced Cx43 protein was detectable by 4–8 h with maximal expression achieved ~24 h after treatment with 1 µg/ml Dox (data not shown). This expression was stable for a minimum of 6–8 days when maintained in medium containing Dox; after removal of Dox a decrease to basal uninduced levels occurred within 24 h.

Immunofluorescent localization of induced Cx43
As shown in Figure 4Go, induced cultures of HIC100 and HIC122 demonstrated punctate Cx43-immunoreactive plaques, consistent with gap junctions, localized to regions of contact between adjacent cells (Figure 4B and DGo), whereas uninduced cultures demonstrated only non-specific background reactivity (Figure 4A and CGo). Increased nuclear and cytoplasmic staining was also seen in induced cultures, presumably resulting from the high levels of Cx43 achieved. Similar results were observed with HIC89 (data not shown). As expected, Dox treatment of the HeLa-On parental line resulted in no increase in immunoreactivity (data not shown).



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Fig. 4. Induced Cx43 is assembled into gap junction plaques as detected by immunofluorescence. Uninduced and induced cultures were grown to confluence and Cx43 was detected by indirect immunofluorescence. Immunofluorescent photomicrographs: (left) uninduced; (right) induced. (A and B) HIC100; (C and D) HIC122. Note the punctate immunoreactivity in the induced cultures in contrast to the low background in uninduced clones. Similar results were seen for HIC89. Only background reactivity was seen in the HeLa-On parental cultures treated and untreated with Dox (not shown).

 
Induction of GJIC in inducible-Cx43 HeLa lines
The functionality of the immunopositive gap junction plaques observed in homologous culture was confirmed by the scrape-loading and microinjection techniques. As seen in Figure 5Go (right), scrape-loading of induced HIC89 (Figure 5AGo) and HIC100 (Figure 5BGo) cells resulted in transfer of dye between adjacent communicating cells whereas uninduced HIC89 and HIC100 cells were incapable of transfer (Figure 5Go, left). Consistent results were seen with HIC122 cultures (data not shown). No dye spread was observed in Dox-treated or untreated HeLa-On cultures (data not shown). In addition, microinjection of membrane-labeled (DiO) HeLa cells seeded onto C3H10T1/2 fibroblast monolayers revealed immediate dye transfer among induced HIC100 and HIC122 HeLa cells, in contrast to the lack of obvious transfer between uninduced HIC100 and HIC122 cells (data not shown). No significant homologous dye transfer was observed with either technique for Dox-treated or untreated parental HeLa-On cells, indicating that the low level of endogenous Cx43 expression in these cells is insufficient for detectable GJIC within the sensitivity of these techniques.



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Fig. 5. Induced cultures exhibit functional homologous GJIC. Communication was detected in high density cultures by the scrape-loading technique. Photomicrographs illustrate the transfer of Lucifer yellow from cells adjacent to the incision to cells distal to the incision in the induced cultures versus a lack of spread in uninduced cultures. Similar results were seen for HIC122. (Left) Uninduced; (right) induced. (A) HIC89; (B) HIC100. No dye spread was observed in HeLa-On line treated and untreated cultures (data not shown).

 
Logarithmic growth and saturation density is unaffected by Cx43 induction
Figure 6AGo shows the logarithmic growth of induced and uninduced HeLa clones (HIC89, 100, 114 and 122) and the HeLa-On parental line. No significant differences in logarithmic growth rates were evident for induced cultures compared with uninduced cultures or in treated versus untreated parental HeLa-On cells. Small differences in total cell number may reflect clonal variations in seeding efficiency. Thus, under these conditions of limited cell–cell contact all clones grew at equivalent rates irrespective of Cx43 induction. Likewise, when HeLa clone HIC122 was seeded at a higher density and allowed to reach confluence where cell–cell interactions are maximal, final saturation densities were not influenced by Cx43 induction (Figure 6BGo). This was also seen with HIC89, HIC100 and HIC114 (data not shown). Alterations in serum levels (1–10%), as well as pre-induction with Dox prior to cell seeding, also resulted in no observed differences in saturation densities with these four cell lines (data not shown). The absence of change in any of these in vitro growth characteristics following treatment with Dox demonstrates the lack of any non-specific effects of the inducing antibiotic.



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Fig. 6. Cx43 induction does not alter growth at low or high cell densities. (A) Low density cultures. (B) High density cultures. Solid symbols, untreated controls; open symbols, 1 µg/ml Dox continuous treatment; •, {circ}, HeLa-On; {blacktriangledown}, {triangledown}, HIC89; {blacksquare}, {square}, HIC100; {blacklozenge}, {lozenge}, HIC114; {blacktriangleup}, {triangleup}, HIC122. Values represent means ± SD of duplicate plates. This experiment was repeated three times with similar results.

 
The possibility remained that Cx43 induction may alter the dynamic equilibrium between cell death and cell proliferation in confluent cultures which would be masked if cultures maintained equivalent saturation densities. To test this hypothesis, uninduced and induced HIC122 cultures were seeded near saturation density, pulse labeled with radioactive thymidine and quantified by scintillation counting to yield tritiated thymidine incorporation per 106 cells. Results demonstrated that an equivalent percentage of cells were proliferating in the induced and uninduced cultures, indicating no dynamic change in proliferation or cell death/detachment (data not shown).

Induced HeLa lines show no alteration in growth control when in contact with non-transformed, growth-controlled fibroblasts
To investigate the response of the inducible HeLa lines to growth regulatory influences from growth-controlled cells, uninduced and induced cultures were seeded onto confluent C3H10T1/2 mouse or NHF monolayers. As shown in Table IGo, the ability of induced clones to form foci was not altered by the presence of the mouse C3H10T1/2 monolayer when compared with uninduced cultures. Likewise, when seeded onto primary human fibroblast monolayers, induced cultures formed equivalent numbers of foci when compared with uninduced cultures (Table IGo). Treatment of cultures for 7 days with agents previously shown to enhance heterologous GJIC (10–8–10–5 M Forskolin and 10–8–10–4 M RO20-1724 cAMP phosphodiesterase inhibitor) (29,47) did not alter focus formation in the presence or absence of Cx43 induction on either monolayer type (data not shown). Experiments utilizing various serum concentrations (1, 2.5 and 5%) and HeLa cultures pre-induced with Dox for 48 h prior to being seeded onto monolayers also revealed no influence of Cx43 induction on focus formation (data not shown).


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Table I. Induced HeLa lines are not growth inhibited by growth-controlled fibroblastsComparison of foci formation of 200 HeLa cells in solo culture (empty dishes) and in co-culture seeded onto confluent mouse (C3H10T1/2) or NHF monolayers under induced (1 µg/ml Dox) and uninduced conditions. Data presented as the means of duplicate dishes ± SD with foci formation in uninduced cultures normalized to 100%. These experiments were repeated three times with qualitatively similar results.Table II. Cx43 induction decreases anchorage-independent growth capacity of HeLa linesHeLa-On, HIC100 and HIC122 lines were treated with 2 µg/ml Dox for 48 h, seeded at low density into semi-solid agarose and maintained for 4 weeks. Colonies were detected microscopically by staining with NitroBlue tetrazolium vital dye. Percent colony number is a ratio of colony formation of induced versus uninduced cultures. This experiment was repeated three times with qualitatively equivalent results.
 
To eliminate the possibility of differences in seeding efficiency between the induced and uninduced cultures, Dox-treated and untreated cells were labeled for 24 h with radioactive thymidine to evaluate plating efficiency. Cell suspensions containing a known number of cells and a known amount of radioactivity were seeded onto confluent monolayers of C3H10T1/2 fibroblasts or empty dishes in the presence or absence of Dox. After 16 h to allow attachment, cultures were harvested, lysed and radioactivity was determined by scintillation counting. No differences in attachment of Dox-treated or untreated HIC122 and HeLa-On cultures were detected (data not shown). Both lines seeded at a lower efficiency in empty dishes (50–60%).

To determine if induced HeLa clones were actually communicating via GJIC with the fibroblast monolayer in co-culture, membrane-labeled (DiO) induced and uninduced HeLa cells were microinjected with Lucifer yellow and the spread of dye to adjacent monolayer fibroblasts was monitored. While homologous GJIC between labeled HIC122 cells was detected as dye transfer between HeLa cells in the induced cultures in comparison with an absence of transfer in the uninduced cultures, neither induced nor uninduced HIC122 or HIC100 cells were capable of heterologous GJIC with monolayer fibroblasts within the sensitivity level of the microinjection technique (data not shown). In addition, treatment of cultures with the cAMP phosphodiesterase inhibitor RO20-1724 (10–8–10–4 M) for 48 h did not result in heterologous GJIC in the uninduced or induced situation for these two clones (data not shown).

Cx43 induction reduces anchorage-independent growth
To investigate the ability of Cx43 expression to alter one index of neoplastic potential, growth capacity in soft agar was quantified. While the ultimate colony sizes were similar, the total number of viable colonies produced by induced HIC100 and HIC122 cultures was decreased by ~50% when compared with uninduced cultures (Table II). No change in colony formation efficiency occurred with the HeLa-On parental line in the presence of Dox. These experiments have been repeated three times with similar results.

In addition, indirect immunofluorescent detection of Cx43 expression in established colonies removed from soft agar cultures indicated that while areas of Cx43 immunoreactivity were detected in small groups of cells in the colony, indicating successful Dox induction of Cx43, the majority of cells were negative for Cx43 expression. This implies the presence of a negative selective pressure in this anchorage-independent environment for cells expressing Cx43.

Cx43 induction attenuates tumorigenicity of HeLa lines in immunodeficient mice
To evaluate the effect of Cx43 expression and GJIC on tumor growth in vivo, immunodeficient (nude) mice were injected with Dox-treated or untreated control HIC100 and HIC122 cells with induction being maintained in vivo by supplying Dox in the drinking water. Parental HeLa-On cells were injected as negative controls. As shown in Figures 7 and 8GoGo, Cx43 induction resulted in a mean decrease in tumor volume of ~50% for both inducible cell lines when compared with uninduced tumors. While the parental HeLa-On cells, in the presence or absence of Dox, and the uninduced clones HIC100 and HIC122 were fully tumorigenic and capable of forming large tumors in vivo, induced clones HIC100 and HIC122 exhibited greatly decreased tumor sizes (Figures 7 and 8GoGo). Figure 7Go shows representative mice from the HIC122 and HeLa-On groups after unilateral injections of control parental HeLa-On line or HIC122 clones in the uninduced (–Dox, Figure 7BGo) and induced (+Dox, Figure 7CGo) situations or mice with bilateral injections of HIC122 cells alone under uninduced or induced conditions (Figure 7AGo). Similar results were observed for the HIC100-injected mice (not shown). The HeLa-On cells, which were unilaterally injected into the same Dox-treated mice as the HIC100 and HIC122 clones, formed tumors which showed no significant alteration in tumor volume when compared with untreated animals (data not shown), again showing a lack of effect of Dox in the absence of the inducible Cx43 construct (Figure 7CGo). Statistical analysis of the tumor volume growth rates revealed highly significant differences between induced and uninduced mean tumor volumes for HIC100 and HIC122 clones (P = 0.0128 and 0.0088, respectively). Dox did not alter the growth rates of the HeLa-On parental cell tumors (P = 0.5959).



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Fig. 7. Induction of Cx43 in HeLa lines attenuates tumorigenicity in immunodeficient mice. Photographs were taken 25 days after bilateral s.c. injection into nude mice. The experiment was terminated at this time because of the large size of the uninduced tumors. (A) Mice injected with HIC122 cells. The two mice on the left received sucrose/water only; the two mice on the right received Dox in sucrose/water. Note the smaller, and in two cases virtually absent, tumors in the Cx43-induced Dox group. (B and C) Mice injected with HeLa-On parental cells in the right flank and HIC122 cells in the left flank (B, untreated; C, Dox-treated). Note the large tumor volumes produced by both cell lines in the non-induced mouse (B), the major reduction in growth of the Cx43-expressing cells in the Dox-induced situation (C) and the lack of Dox effect on growth of the HeLa-On parental line (C). Mice shown are representative and similar results were obtained with the HIC100 line (not shown). Arrows denote tumor sites.

 



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Fig. 8. Cx43 induction reduces the growth rate of s.c. injected HeLa lines HIC100 and HIC122 in immunodeficient mice. Tumor sizes were measured in two dimensions every 3–4 days and volumes calculated assuming spherical shape (mm3). (A) HIC100; (B) HIC122. {circ}, Induced (+Cx43); •, uninduced. Numbers by data points represent total number of tumors detected over total number of injections for each corresponding clone. Only detectable tumors were included in mean tumor volume calculations. Statistical analysis revealed highly significant differences between induced and uninduced mean tumor volumes in the HIC100 and HIC122 clones (P = 0.0128 and 0.0088, respectively) with no significant difference observed between the HeLa-On parental cell tumors from untreated and Dox-treated mice (P = 0.5959).

 
Inducible expression of exogenous Cx43 in vivo
Total RNA was isolated from excised tumors resulting from parental HeLa-On, HIC100 and HIC122 injections (Figures 7 and 8GoGo) and analyzed by northern blotting for in vivo regulation of the exogenous Cx43 gene. Autoradiographs were digitally quantified and normalized to GAPDH transcript levels (data not shown). A mean 1.7-fold increase in production of the 2.2 kb exogenous Cx43 transcript was detected in four HIC122 tumors from Dox-treated mice when compared with untreated mice. We detected no increased expression in four HIC100 tumors or the parental HeLa-On tumors from Dox-treated mice (data not shown).

Portions of these tumors were fixed with formalin, embedded in paraffin, sectioned and subsequently tested for Cx43 protein production by immunofluorescent analysis as before. As shown in Figure 9Go, small areas of punctate immunoreactivity were detected in Dox-treated tumors derived from HIC100 (Figure 9BGo, bottom) and HIC122 cells (Figure 9DGo, bottom), whereas no such areas were observed in tumors derived from HIC100 or HIC122 cells from untreated mice (Figure 9A and CGo, bottom) or the parental HeLa-On line from Dox-treated mice (data not shown). However, the majority of the tumor mass from both inducible cell lines (Dox-treated mice) was devoid of immunoreactive plaques, strongly suggesting clonal selection of non-inducible cells. Lack of Cx43 expression in HeLa cells was seen in regions surrounding blood vessels, indicating that lack of induction was not a consequence of insufficient Dox availability.



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Fig. 9. Immunofluorescent detection of Cx43 expression in vivo. Paraffin-embedded tumor sections were detected by indirect immunofluorescence for Cx43 protein as in Figure 4Go. H&E stained sections (top), phase photomicrographs (middle) and immunofluorescent photomicrographs (bottom). The middle and bottom panels represent the same field of tumor sections. (A) Uninduced HIC100 (– Dox); (B) induced HIC100 (+ Dox); (C) uninduced HIC122 (– Dox); (D) induced HIC122 (+ Dox). Note areas of punctate immunoreactivity in induced tumors only.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To our knowledge, these studies are the first to describe a loss of Cx43 expression in pre-malignant lesions of the cervix, indicating that this is a very early event in the carcinogenic process. Since most of these lesions can be expected to be positive for HPV (human papilloma virus) (48,49), it is unclear whether this loss is a direct consequence of viral infection or is secondarily involved in the carcinogenic process. However, our demonstration that loss of expression is also seen in dysplastic regions of the oral cavity (50), where exposure to tobacco products is the main etiological agent, suggests that this is a common event in epithelial preneoplasia. Multiple lines of evidence suggest that this loss of Cx43 expression will be accompanied by increased proliferation and thus lead to an acceleration of the process of carcinogenesis. The relationship between connexin expression and proliferation is clearly evident from the present study in which Cx43 expression decreased the growth rate of HeLa cells in two assays predictive of neoplasia (Figures 7 and 8GoGo and Table II). Unfortunately, for practical reasons, we were not able to conduct such studies utilizing dysplastic cells in which neoplastic progression was not so advanced. In this situation, we believe that Cx43 expression would have had more pronounced effects on growth control. In normal human skin, Cx43 expression can be up-regulated by all-trans retinoic acid (ATRA), and we predict that this would also occur in oral and cervical epithelium, sites at which ATRA possesses cancer chemopreventive activity (30,51). It is an interesting speculation, which we are currently testing, that ATRA may also increase Cx43 expression in dysplastic cells and reduce their proliferation. The association between dietary carotenoid intake and decreased risk of cervical carcinoma (31,32) could also in part be explained by the demonstrated ability of major dietary carotenoids to increase Cx43 expression in cultured human keratinocytes (52). Thus, Cx43 may play a role as a tumor suppressor gene and may also prove to be a useful intermediate marker of response to certain classes of cancer preventive agents.

Our development of HeLa cell lines containing an inducible Cx43 gene has allowed for the first time the evaluation of the effects of Cx43 induction both in vitro and in vivo. Because these inducible cells can serve as their own controls when not induced, the influence of gene expression can be determined unambiguously. Moreover, as we have shown in vitro and in vivo, Dox itself has no effect on the phenotypic behavior of HeLa cells which express only the rtTA protein (HeLa-On) (Figures 6–8GoGoGo), demonstrating that these effects rely on the inducible gene. In inducible cells, the addition of Dox results in high levels of Cx43 mRNA and protein production within 24–36 h (Figures 2 and 3GoGo). This Cx43 is processed and assembled into immunoreactive junctional plaques (Figure 4Go) resulting in the restoration of homologous GJIC (Figure 5Go), as has been shown previously in HeLa cells after transfection with a Cx43 expression construct driven by constitutive promoters (33,34). Logarithmic growth rates of all four clones analyzed were not influenced by expression of Cx43 (Figure 6AGo). This is not surprising as cell–cell interactions are at a minimum at these low cell densities. However, even at high cell densities, when cell–cell interactions were extensive, Cx43 induction did not influence saturation density (Figure 6BGo) or reduce proliferation (data not shown). In contrast, Cx43 expression reduced two indicators of the neoplastic potential of these cells: the ability to grow in an anchorage-independent environment (Table II), a property associated with human tumor stem cells (53); the ability to grow as tumor xenografts (Figures 7 and 8GoGo). That these responses are not due to possible non-specific effects resulting from overexpression of the introduced gene is indicated by the lack of response of these cells to Cx43 induction when in monolayer culture (Figure 6Go). Others have reported on the failure of Cx43 induction (5456) or inhibition using antisense constructs (57,58) to influence proliferation rates of several tumor cell lines and, consistent with the present studies, that inhibition of Cx43 expression results in enhanced tumorigenicity (58). Transfection of the connexin32 gene into C6 glioma and SKHep1 cell lines also showed a differential effect: attenuation of tumor growth in vivo was seen with no measurable effect on growth in vitro (54,55). However, our results contrast with other studies, in which transfection of connexin genes driven by constitutive promoters was reported to enhance growth control in vitro (5962). The present results also are in contrast to another study utilizing HeLa cells where expression of connexin26, but not Cx43, was shown to influence in vivo tumorigenicity (33). Whether these variations in results reflect differences in the lineage or derivation of the cells studied or, as discussed below, are the result of confounding effects due to unrecognized clonal heterogeneity remains to be determined. However, it is now clear that Cx43 can indeed influence the neoplastic phenotype of HeLa cells.

The ability to rigorously examine the effects of conditionally expressing one specific gene is particularly crucial in studies involving tumorigenicity in vivo where multiple factors can influence tumor growth. Without this control, it is difficult to separate effects of expression of the gene of interest on measured parameters from the influence of pre-existing differences, present within a population of cells, which are only revealed after clonal selection of antibiotic-resistant cells following transfection of the population. We first became aware of the issue of clonal heterogeneity when we discovered significant heterogeneity in growth characteristics between subclones of C3H10T1/2 cells (unpublished results). We again encountered this problem during studies utilizing HeLa cells obtained from the ATCC, where clones derived from the original HeLa cell line (CCL-2) exhibited high endogenous Cx43 expression levels which correlated with decreased saturation densities in vitro as well as diminished tumorigenicity in vivo (63). Thus, selection even in the absence of gene transfection can give rise to cells with highly diverse phenotypes.

The lack of influence of Cx43 expression on growth rates in monolayer culture indicates that the cells are unable to generate and/or respond to homologous junctional signals. We have previously determined that agents which elevate cAMP levels can induce GJIC between growth-controlled normal and transformed cells and that induction is statistically associated with growth arrest of the transformed cells (29). To determine if these HeLa cells were capable of responding to the influence of normal cells, induced or uninduced HeLa cells were cultured with non-transformed mouse (C3H10T1/2) or normal human fibroblasts, both of which normally express Cx43 (22,64). Induction of Cx43 resulted in homologous GJIC between HeLa cells, however, we could not detect heterologous GJIC between HeLa cells and fibroblasts nor did co-culture decrease the ability of HeLa cells to form foci (Table IGo). However, this lack of detectable heterologous GJIC leaves open the possibility that these HeLa lines may still possess the ability to respond to GJIC-mediated growth-controlling signals from normal cells if barriers to heterologous GJIC were removed.

That Cx43 can alter the neoplastic potential of these HeLa cells was evident in two assays previously shown to be indicators of neoplasia (Figures 7 and 8GoGo and Table II). Cx43 induction strongly diminished the number but not the size of anchorage-independent colonies which formed in soft agar. In induced cultures, many small colonies, too small to score, were observed.

Immunofluorescent staining of large colonies from induced cultures revealed that while Cx43 was expressed in islands of cells, most cells were negative for Cx43 expression. As discussed below, this is the situation we discovered in tumor xenografts. That reductions in growth potential were not seen under adherent growth conditions (Table IGo and Figure 6Go) indicates that Cx43 expression specifically reduces the neoplastic potential of these HeLa cells. Whether this action is due to GJIC between cells in the small colonies seen in the induced cultures or to other properties of Cx43 remains to be determined. A similar differential response was observed in reported studies involving inhibition of insulin-like growth factor-1 receptor expression in human melanoma cells. Inhibition decreased cell survival in vivo and reduced anchorage-independent growth but did not reduce monolayer growth (65).

Reduced neoplastic potential was also seen following Cx43 induction in the mouse xenograft studies. As Dox did not influence the ability of the control HeLa-On line to form tumors, this demonstrated that Dox itself is not responsible for the effects seen (Figures 7 and 8GoGo). This was most dramatically exhibited in the Dox-treated mice which had one flank injected with the HeLa-On parental cells and the other flank with inducible HIC100 or HIC122 cells. As in the anchorage-independent growth studies, while Dox treatment did attenuate tumor growth, it did not completely abolish the neoplastic potential of these cells. Studies of excised tumors indicate a reason for this partial response: northern analysis of RNA extracted from Dox-treated HIC122 tumors indicated only a small increase (1.7-fold) in exogenous Cx43 expression over non-induced tumors; an increase much less than observed in cultured cells (Figure 3Go). Moreover, immunofluorescent detection of Cx43 in tumor sections demonstrated that while nests of Cx43 immunopositive cells were seen in Dox-treated but not control tumors (Figure 9Go), a majority of cells within the induced HIC100 and HIC122 tumors were deficient in Cx43 protein. We thus conclude that those tumors growing in Dox-treated mice resulted predominantly from selection of cells which had lost Cx43 inducibility. This loss in the presence of a strong selection pressure is perhaps not surprising as the majority of human tumors have been shown to display chromosomal instability, presumably due to various mutations in genes responsible for chromosomal maintenance and segregation (66). It seems likely that HeLa cells would be even less able to simultaneously maintain two exogenously introduced constructs in their genome, resulting in the observed loss of Cx43 gene inducibility. Our data do not allow us to determine whether this loss of inducibility occurred before or after injection of these cells into the mice. Certainly, the immunofluorescent studies conducted in vitro (Figure 4Go) gave no indication of heterogeneity on the scale seen in the tumors (Figure 9Go) and the comparable findings in the soft agar assays (data not shown) would suggest that loss of inducibility can occur over a period of 4 weeks. We would hypothesize that were it possible to avoid such loss of inducibility, tumor growth rates would have been even lower until cells evolved additional mechanisms to avoid growth control.

The present results, for the most part, support the concept of GJIC influencing growth control as first proposed by Loewenstein (37,38). However, the apparent lack of effect of Cx43 induction in monolayer cultures is puzzling. As connexins can function both as conduits for intercellular signals and potentially as transmembrane cell–cell receptors, these HeLa cell lines may provide a tool by which to separate these two aspects of connexin function. Thus, the influence of GJIC in enhancing in vitro growth control of transformed C3H10T1/2 cells (67) and early passage HeLa cells (63) may be a consequence of direct junctional transfer of growth regulatory signals to cells in which the ability to respond to such signals is still intact. In contrast, the influence of connexin expression on aspects of the neoplastic phenotype, i.e. anchorage-independent growth and in vivo tumorigenicity, as seen here and in other established tumor lines, may be a consequence of other functions of connexins, perhaps in conjunction with cell–cell adhesion molecules (68). In summary, with respect to these inducible-Cx43 HeLa cell lines, restoration of Cx43 expression influences the more advanced aspects of the neoplastic phenotype, specifically anchorage-independent growth in vitro and tumorigenicity in vivo.


    Notes
 
5 Present address: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA Back

6 To whom correspondence should be addressed Email: john{at}crch.hawaii.edu Back


    Acknowledgments
 
We acknowledge the assistance of K. Jeraj, S. Case, K. Newkirk and L. Magee of the University of Hawaii Laboratory Animal Services in the nude mouse study and also of the CRCH Statistics Core Resource with statistical analysis. Thanks are due to S. Reed (La Jolla) and the H. Bujard laboratory (Germany) for the pUHD10-3 plasmid construct and E. Beyer (St Louis) for the pG2A-Cx43 plasmid construct. This work was supported by NIH grant CA74669 and a grant from the USDA. T.J.K. was the recipient of an Institutional NIH-T32 Research Training Grant and a Meji Foundation Fellowship and K.A.S. was the recipient of a Howard Hughes Medical Institute Undergraduate Fellowship.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 14, 1999; revised December 2, 1999; accepted December 20, 1999.