Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression

Timothy J.King1,2, Laurie H.Fukushima1, Timothy A.Donlon3, A.David Hieber4, Kelly A.Shimabukuro1 and John S.Bertram1,3,5

1 Molecular Carcinogenesis, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI 96813 and
2 Cell, Molecular and Neurosciences Program, Department of Genetics and Molecular Biology,
3 Department of Genetics and Molecular Biology and
4 Department of Plant Molecular Physiology, University of Hawaii at Manoa, Honolulu, HI 96822, USA


    Abstract
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 Abstract
 Introduction
 References
 
A HeLa cell line, obtained from the ATCC, was cloned and found to exhibit a spectrum of in vitro and in vivo growth characteristics as well as variable expression of endogenous connexin43 (Cx43), a widely expressed gap junction protein implicated in growth control. The majority of clones expressed functional Cx43, which contrasted with previous studies reporting that HeLa cells are completely negative for Cx43 mRNA/protein expression. This endogenous Cx43 expression correlated with increased growth control: Cx43-positive clones exhibited a decreased saturation density and a diminished growth capacity when in co-culture with growth-controlled normal cells in constrast to Cx43-negative clones. Endogenous Cx43 expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth and decreased tumorigenicity in immunodeficient mice. Treatment of Cx43-negative cells with 5-aza-2'-deoxycytidine resulted in expression of Cx43, suggesting gene silencing via DNA methylation. These results support the concept of growth control via junctionally transmitted signals and suggest an epigenetic mechanism for tumor cells to circumvent this control during carcinogenesis. Moreover, the heterogeneous nature of this cell line and the ease of connexin43 gene induction suggest caution in the interpretation of results involving gene transfection using noninducible gene expression systems.

Abbreviations: 5-aza-CdR, 5-aza-2'-deoxycytidine; Cx43, connexin43; GJIC, gap junctional intercellular communication.


    Introduction
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 Abstract
 Introduction
 References
 
The HeLa cell line, which was derived from a human cervical carcinoma in the 1950s, has been extensively used as a tool for examining the cellular and molecular biology of human neoplasia due to the relative ease of cell cultivation, resulting from high viability and rapid growth, as well as being amenable to molecular manipulations such as gene transfer. HeLa cells have been widely used in studies designed to determine the effects of increased expression of exogenous genes on cell behavior and these types of studies have extended to examination of the influence of connexin gene expression on growth control and neoplastic behavior (17). Interpretation of results from these connexin gene transfer studies has been simplified by reports that HeLa cells lack endogenous expression of several different connexins (3,8). Connexins are a family of at least 14 highly conserved proteins which demonstrate developmental and tissue-specific expression patterns and, when integrated as a hexamer into the plasma membrane, form the functional unit of a connexon or hemi-channel (2,9,10). Hemi-channels from two neighboring cells can dock to form a gap junction channel which allows ions and hydrophilic molecules <1–2 kDa in size to traverse from one cell to another via gap junctional intercellular communication (GJIC) (1,11,12). Connexins and GJIC have been implicated in regulation of cell growth and differentiation (1,9).

One of the problems in the interpretation of gene transfer studies such as those mentioned above, involving the stable integration of an exogenous gene where expression is controlled by a constitutive promoter, is that because of the extensive selection required following transfection, i.e. selection for antibiotic resistance as well as expression of the gene of interest, chosen cells may differ in many respects from those cells initially transfected. If the original population is heterogeneous, selection can result in a new population whose properties are skewed from the mean of the original population; if the original population is genetically unstable, as is the case with most tumor cells (13), a genetic change early in the selection process could result in a population which differs markedly from that present originally, a phenomenon equivalent to the so-called founder effect in human populations (14). These problems of clonal heterogeneity can be overcome by the use of inducible gene expression systems. Recently, Gossen and Bujard described the development in HeLa cells of a tetracycline-responsive mammalian gene expression system, based on the bacterial tetracycline receptor–HSV VP-16 transactivator fusion protein (tTA) and tetracycline receptor operator sequences (tetO), where exogenous gene expression can be rapidly regulated by the antibiotic tetracycline (15,16).

In order to engineer a tetracycline-inducible connexin43 (Cx43) cervical carcinoma cell line, HeLa cells only two passages after receipt from the ATCC (CCL-2, batch F-12958) were stably transfected with a plasmid containing a tetracycline-responsive trans-activator (tTA) gene and a neomycin (G418) resistance gene (pUHD15-1) resulting in G418-resistant clones (16,17). A total of 44 colonies were isolated and preliminarily analyzed for background endogenous Cx43 expression by western immunoblotting. As shown in Figure 1Go, while a few clones were negative for Cx43 expression, the majority of these clones (42/44, 95%) expressed Cx43, with many demonstrating high levels (10/44, 23%). These clones appear stable with respect to Cx43 expression as cultures of these clones analyzed 40 passages later by western immunoblot exhibited no decrease in Cx43 expression levels (data not shown). It should again be stressed that these phenotypic differences existed in the absence of any transfected exogenous Cx43 expression construct. HeLa clones were evaluated by northern blotting (data not shown) with clones positive for endogenous Cx43 protein exhibiting a single prominent band at ~3.1 kb, as would be expected for endogenous human Cx43 mRNA transcripts, whereas the Cx43-negative clones exhibited no detectable hybridization in this region (18).



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Fig. 1. Spectrum of endogenous Cx43 protein expression among HeLa clones. A total of 44 G418-resistant clones were analyzed by 10% SDS–PAGE and chemiluminescent detection protocols (Tropix) using a polyclonal antibody directed against amino acids 368–382 of rat Cx43 (36). Autoradiographs were quantified with NIH Image software and expressed in arbitrary units. (Inset) Cx43 protein expression in G418-resistant HeLa clones. Protein lysates (40 µg protein/lane) from five representative clones (Cx43-positive, 1, 36 and 87; Cx43-negative, 54 and 70) were analyzed by western blotting protocols. Lane 1, clone 1; lane 2, clone 36; lane 3, clone 54; lane 4, clone 70; lane 5, clone 87.

 
To address the possibility that the transfection protocol or the stably integrated tTA-expressing construct either altered endogenous Cx43 expression or targeted a select subpopulation of HeLa cells, we cloned the original ATCC HeLa population by low density seeding without transfection or selection procedures. Western analysis of 44 clones produced results similar to those seen with the G418-resistant clones: 98% (43/44) of these clones expressed endogenous Cx43, a figure comparable to the 95% positive expression seen in the G418-resistant clones (data not shown). These non-transfected clones were not utilized further, instead, all biological data were obtained using the G418-resistant clones.

Cx43-expressing HeLa clones were capable of assembling Cx43 protein into immunoreactive gap junction plaques, localized to regions of contact between adjacent cells, as determined by indirect immunofluorescence (data not shown). These immunoreactive gap junction plaques were capable of GJIC, as observed in homologous culture detected by the scrape loading technique (19) as well as by the pre-loading calcein dye transfer technique, whereas Cx43-negative clones were incapable of dye transfer (data not shown). While these clones were not examined for expression of any other connexin types, lack of dye transfer in Cx43-negative clones argues against significant expression levels of other connexins.

Because previous reports had indicated that HeLa cells are negative for endogenous Cx43 (3,8), we confirmed the identity of these HeLa clones. As expected, karyotypic analysis (20) revealed that all clones tested (clones 1, 36, 54, 70 and 87) contained human chromosomes, were negative for the presence of the Y chromosome, exhibited the expected modal chromosomal numbers and diagnostic HeLa marker chromosomes in addition to glucose 6-phosphate dehydrogenase alloenzyme type A (G6PD) (Innovative Chemistry Inc.) and HPV-18 sequences consistent with ATCC information and previously published results for the HeLa cell line (data not shown) (21,22).

Analysis of the logarithmic growth of eight HeLa clones representing a range of endogenous Cx43 expression, from no Cx43 (clones 54 and 70), to low (clones 5, 64, 67 and 74) to high (clones 1 and 78), demonstrated no significant difference between the clones. Thus, under conditions of limited cell–cell contact, all clones grew at equivalent rates irrespective of Cx43 expression (data not shown). In contrast, when clones were seeded at a higher density and allowed to reach confluence, where cell–cell interactions are maximal, clones expressing Cx43 (clones 1, 36 and 87) exhibited significantly lower saturation densities than those clones lacking Cx43 (clones 54 and 70) (Figure 2AGo). The mean saturation density of Cx43-positive clones (1, 36 and 87) was 2.3x106 cells/60 mm dish, while that for Cx43-negative clones (54 and 70) was 3.1x106 cells/60 mm dish, an increase of 35%. This increased growth advantage of Cx43-negative clones may explain the discrepancy between these results and previously published studies documenting the absence of Cx43 expression in HeLa cell lines as Cx43-negative cells can be expected to dominate cultures after continuous passaging (3,8).



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Fig. 2. Growth of Cx43-positive HeLa clones differs from Cx43-negative clones at high cell densities and in anchorage-independent environments. (A) Growth curves of HeLa clones (Cx43-negative, 54 and 70; Cx43-positive, 1, 36 and 87) in late logarithmic and saturation phase growth (seeded at 5x105 cells/60 mm dish) are presented as the mean of duplicate plates ± SD. Statistical analysis by t-test revealed a significant difference in final densities between Cx43-positive clone 1 and both Cx43-negative clones 54 (P < 0.024) and 70 (P < 0.019). (B) Anchorage-independent growth of HeLa clones inversely correlates with Cx43 expression. Cells were seeded at low density (200 cells/60 mm dish) in semi-solid agarose (0.4%, Type II; Sigma) and maintained for 4 weeks. Colonies were visualized by addition of NitroBlue Tetrazolium vital dye (1 mg/ml; Sigma) and measured microscopically. Volume was calculated on the assumption of a spherical colony shape.

 
To investigate the neoplastic potential of these clones, the ability of the cells to form colonies in soft agar was quantified. While the total number of viable colonies was similar between clones, the sizes of the individual colonies were much smaller for Cx43-positive clones (1, 36 and 87) when compared with colony sizes achieved by Cx43-negative clones (54 and 70) (Figure 2BGo). These data indicate that cell density is a major factor limiting anchorage-independent growth for Cx43-expressing HeLa clones.

In addition, the ability of Cx43-positive HeLa clones to form foci was greatly diminished in the presence of a confluent mouse (C3H10T1/2) (Table IAGo) or human fibroblast monolayer (Table IBGo) in comparison with empty culture dishes, implicating Cx43 expression in the transduction of growth-controlling signals derived from neighboring cells (23). In contrast, focus formation by those clones lacking Cx43 was enhanced by the presence of the mouse fibroblast monolayer when compared with cells plated in empty dishes. These results were not due to unequal seeding of monolayers in comparison with empty dishes, as experiments utilizing HeLa clones radiolabeled with thymidine demonstrated that all clones exhibited a higher seeding efficiency on mouse monolayers in comparison with seeding in empty dishes. However, microinjection of Lucifer yellow dye into Cx43-positive and Cx43-negative clones after seeding onto mouse fibroblast monolayers revealed no heterologous dye exchange (data not shown). This lack of dye transfer between epithelial and fibroblastic cell types has previously been reported and may explain these results (24).


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Table I. Growth inhibition of HeLa clones by confluent, growth-controlled fibroblast monolayers
 
To evaluate the effect of Cx43 expression and GJIC on tumor growth in vivo, immunodeficient nude mice were injected with Cx43-positive clones (1 and 36) or Cx43-negative clones (54 and 70) followed by tumor measurement twice weekly (Figure 3Go). Both Cx43-negative clones formed more rapidly growing tumors than did the Cx43-positive clones, implicating Cx43 expression in modulation of growth in vivo.




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Fig. 3. Reduced tumorigenicity of Cx43-positive cells in vivo. Immunodeficient mice (Crl:CD-1-nuBR; Charles River Laboratory) were injected bilaterally into each flank with 3x106 cells. Representative mice are shown 75 (clones 36 and 54) and 91 days (clones 1 and 70) post-injection. (A) (left) Two mice, bilateral injection of clone 36 (Cx43-positive); (right) two mice, bilateral injection of clone 54 (Cx43-negative). (B) (Left) One mouse, bilateral injection of clone 1 (Cx43-positive); (right) one mouse, bilateral injection of clone 70 (Cx43-negative). Arrows denote injection sites. (C) Cx43 expression is associated with lower tumor growth rates. Tumor size in mice shown in (A) and (B) was measured every 3–4 days with volumes calculated assuming spherical tumors (mm3). Experiment A (circles): clones 36 (Cx43-positive, {circ}) and 54 (Cx43-negative, •); experiment B (triangles): clones 1 (Cx43-positive, {triangleup}) and 70 (Cx43-negative, {blacktriangleup}). Numbers represent total final number of tumors over total number of injections for each corresponding clone. Only detectable tumors were included in mean tumor volume calculations. Cx43-positive clones had similar average tumor growth rates (P < 0.40), however, each of the Cx43-negative clones showed significantly greater tumor growth rates (P < 0.001) when compared with the average of the two Cx43-positive clones.

 
Northern analysis revealed that Cx43-negative clones contained no detectable Cx43 mRNA (data not shown) and karyotypic analysis revealed no gross deletions in the connexin43 gene locus on human chromosome 6 (data not shown), implying some defect in the connexin43 gene itself or in its transcription. To evaluate methylation as a mechanism for connexin43 gene suppression, two clones negative for endogenous Cx43 expression were treated with the nucleoside analog 5-aza-2'-deoxycytidine (5-aza-CdR) and analyzed for expression of Cx43 by western blotting (Figure 4Go). Cx43-negative clone 54 expressed increasing amounts of Cx43 protein following treatment with increasing concentrations of 5-aza-CdR (Figure 4AGo). Similar results were obtained with a second Cx43-negative clone (clone 70) (Figure 4BGo). This expression was stable in the absence of continued treatment for up to 12 passages (~3 months) (data not shown).



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Fig. 4. Restoration of endogenous Cx43 expression in Cx43-negative HeLa lines by treatment with 5-aza-CdR (26). Cells were treated for 24 h with the indicated concentrations of 5-aza-CdR and harvested after 3 days. Western blots were performed and detected as for Figure 1Go. (A) Western blot. Lanes 1–9, clone 54; lanes 1 and 9, untreated control; lanes 2–8; 5-aza-CdR at 3x10–8, 10–7, 3x10–7, 10–6, 3x10–6, 10–5 and 3x10–5 M, respectively. Lanes 10 and 11, clone 70; lane 10, untreated control; lane 11, 3x10–5 M 5-aza-CdR.

 
While these studies are supportive of the hypothesis of GJIC and growth control, they are only correlative and again stress the need for analysis using inducible gene expression systems. Furthermore, had we transfected a construct containing a connexin43 gene under the control of a constitutive promoter, as has previously been done by others (3,4,6,7), we may have arrived at confusing conclusions as to the role of GJIC in growth control due to pre-existing variations in endogenous Cx43 expression and/or growth control within this line (summary in Table IIGo). Moreover, even among our Cx43-positive and Cx43-negative clones, considerable variation exists in the ability to become growth controlled by quiescent fibroblasts (Table IGo) or to grow in immunodeficient mice (Figure 3Go).


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Table II. Summary of HeLa clone characteristics
 
Treatment of two Cx43-negative clones with 5-aza-CdR resulted in the expression of endogenous Cx43 protein, suggesting that hypermethylation of the connexin43 promoter, or a gene regulating this promoter, is responsible for suppression of Cx43 expression and loss of Cx43-mediated GJIC (Figure 4Go) (26). We have recently subcloned these 5-aza-CdR-treated cells and obtained clones which express Cx43 at levels comparable with that seen with the high expressing clones 1, 36 and 87 (manuscript in preparation). These clones are currently being analyzed for altered growth characteristics to evaluate the influence of Cx43 expression on specific growth parameters. In addition, antisense technology is being employed to evaluate the importance of Cx43 expression on the behavior of these re-expressing cells.

These data indicate a possible general mechanism for loss of Cx43 expression in these cervical carcinoma cells as well as other Cx43-negative epithelial tumors (1). Indeed, studies to determine specific mutations in connexin genes within tumor lines negative for connexin expression have failed to identify mutations or deletions implicating the silencing of connexin gene expression via an alternative mechanism(s) (2729). In support of these observations, altered methylation patterns in the promoters of the connexin43 and connexin32 genes in rat liver cells have been reported (30). It is well established that hypermethylation can result in decreased expression of tumor suppressor genes (p16, p15 and hMLH-1), contributing to increased tumorigenicity in various tumor cell types (3133). Studies to determine the direct influence of gene methylation on Cx43 expression in these HeLa cell lines are currently being performed.

If connexin and GJIC loss is secondarily involved in the carcinogenic process, being permissive but not causative, primary tumors and transformed cell lines, such as HeLa, would be expected, at least initially, to demonstrate a spectrum of phenotypes with respect to loss of connexin expression or GJIC function as shown here. Indeed, the presence of connexin expression and functional GJIC has been documented in many tumor lines (1,9). Importantly, both Cx43-negative and Cx43-positive clones are fully tumorigenic and capable of forming tumors in nude mice, although to differing extents. These results imply a role for connexins and GJIC in modulating but not completely controlling expression of the transformed phenotype. We interpret these findings as evidence that Cx43 expression does not alter the transformed nature of these tumor cells, but strongly influences their behavior. These results imply that loss of GJIC plays a significant role in neoplastic progression; further support for this statement comes from reports of the enhanced susceptibility of connexin32 knockout mice to carcinogenesis of the liver (34), an organ in which connexin32 is extensively expressed. Unfortunately, this question cannot be addressed in connexin43 knockout mice as neonates die shortly after birth due to heart defects (35).


    Acknowledgments
 
Special thanks are due to Brenda Y.Hernandez (CRCH, University of Hawaii) for HPV sequence typing of HeLa clones. We acknowledge the assistance of K.Jeraj, S.Case, K.Newkirk and L.Magee at the University of Hawaii Laboratory Animal Services in the nude mouse study and also of J.Grove (CRCH Statistics Core Resource) in statistical analysis. Thanks are due to the S. Reed laboratory (La Jolla) for the pUHD15-1 (tTA) construct. This work was supported by NIH grant CA74669 and a grant from the USDA. T.J.K. was the recipient of an 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.


    Notes
 
5 To whom correspondence should be addressed at: Molecular Carcinogenesis, Cancer Research Center of Hawaii, Department of Genetics and Molecular Biology, University of Hawaii at Manoa, Honolulu, HI 96813, USAEmail: john{at}crch.hawaii.edu

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Received June 25, 1999; revised August 23, 1999; accepted September 17, 1999.