Retroviral Delivery of Connexin Genes to Human Breast Tumor Cells Inhibits in Vivo Tumor Growth by a Mechanism That Is Independent of Significant Gap Junctional Intercellular Communication

Qin, Hong; Shao, Qing; Curtis, Heather Leigh; Galipeau, Jacques; Belliveau, Daniel J.; Wang, Taiqi; Alaoui‐Jamali, Moulay A.; Laird, Dale W.

doi:10.1074/jbc.m200797200

Cited by 199 publications

(185 citation statements)

References 39 publications

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“…The fs260-GFP construct was further mutated to substitute the putative endoplasmic reticulum FF motif, encoded within the aberrant 46 amino acids, to an AA motif (fs260-FF/AA) by using the forward primer 5Ј-C-GACAGAAACAAGCCGCCTTGCCGCAATTACAACAAGC-AAGCAAG, and reverse primer 5Ј-CTTGCTTGCTTGTTGTA-ATTGCGGCAAGGCGGCTTGTTTCTGTCG. All GFP-tagged variants were further cloned into the AP2 retroviral vector as described previously (25). Transient Transfection and Retroviral Infection-Cells were transfected with human Cx43-GFP, fs260-GFP, T259-GFP, or fs260-FF/AA-GFP by Lipofectamine 2000 (Invitrogen) as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of aGJA1Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma

Gong¹,

Shao²,

Lounsbury³

et al. 2006

J. Biol. Chem.

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A frameshift mutation generated from a dinucleotide deletion (780 -781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.Gap junctions are unique intercellular channels that directly connect the cytoplasm of neighboring cells, which are formed by integral membrane proteins called connexins (Cxs) 2 (1). Each apposing cell contributes a hexameric arrangement of connexins, also known as a connexon, which dock to form a gap junction channel. A connexin polypeptide chain spans the plasma membrane four times, consisting of four transmembrane domains (M1 to M4), two extracellular loops (EL-1 and EL-2) and one intracellular loop (IL), with both the N and C termini (AT and CT, respectively) located in the cytoplasm (2-4) (Fig. 1). Whereas connexin transmembrane domains and extracellular loops are highly conserved, the intracellular loop and C terminus are known to be the most variable domains across different connexin family members, suggesting that these domains play important roles in the differential functions of connexins.Connexins exhibit complex tissue distribution patterns that are temporally and spatially regulated during development and differentiation. Among connexin family members (21 in human and 20 in mouse), the GJA1-encoded protein (Cx43) exhibits the most ubiquitous distribution, playing important roles in many tissue types and physiological processes (5). Cx43 channels can be regulated by a pH-sensitive "ball-and-chain" mec...

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Section: Methodsmentioning

confidence: 99%

Functional Characterization of aGJA1Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma

Gong¹,

Shao²,

Lounsbury³

et al. 2006

J. Biol. Chem.

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“…Therefore, the mechanisms by which Cxs inhibit tumor growth were originally proposed to be through the diffusion of putative growth inhibitory factors via the Cx-modulated GJs (10). However, increasing evidence suggests that the GJ-independent roles are also involved in the Cx-induced growth inhibition (5,6,(11)(12)(13)(14). Further, it was reported that the C-terminal domain showed the same growth inhibition effects as the full-length Cx43 in certain cell lines (13,15) (and the present study).…”

Section: Discussionmentioning

confidence: 99%

The Gap Junction-independent Tumor-suppressing Effect of Connexin 43

Zhang

Kaneda

Morita

2003

Journal of Biological Chemistry

169

128

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The gap junction gene connexin 43 (Cx43) showed tumor-suppressing effects on various tumor cell lines. We have previously demonstrated that Cx43 inhibited expression of S phase kinase-associated protein 2 (Skp2), the human F-box protein that regulates the ubiquitination of p27. Cx43 did not alter the mRNA level of SKP2, but it promoted the degradation of the Skp2 proteins (Zhang, Y. W., Nakayama, K., Nakayama K. I., and Morita, I. (2003) Cancer Res. 63, 1623-1630). In this study, we showed that the specific gap junction inhibitor 18 ␤-glycyrrhetinic acid did not influence the inhibitory effect of Cx43 on Skp2 expression. Further, the deletion mutation analyses demonstrated that the C-terminal domain of Cx43 that did not form gap junctions was sufficient to inhibit expression of Skp2, whereas the N-terminal domain that formed the gap junctions did not show such an effect. Like the full-length Cx43, the Cterminal domain also increased the protein instability of Skp2, whereas the N terminus did not. Moreover, the C-terminal domain was as effective as the full-length Cx43 in inhibiting cell proliferation; however, the Nterminal domain did not show any inhibitory effect on cell proliferation. Therefore, these data revealed a gap junction-independent pathway for Cx43 to inhibit tumor growth by suppressing the Skp2 expression.

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“…Following transfection, replicationdefective retroviral supernatants were collected and filtered through a 0.45-m filter (Pall Gelman Laboratories, Ann Arbor, MI). BICR-M1R k cells expressing Cx43-GFP were engineered to stably express Panx1 and Panx3 by following a previously described protocol (30). Retrovirus encoding Panx1-GFP was also used to stably express GFP-tagged Panx1 in BICR-M1R k cells.…”

Section: Transfection and Engineering Of Stable Cell Lines-dsredtaggementioning

confidence: 99%

Pannexin1 and Pannexin3 Delivery, Cell Surface Dynamics, and Cytoskeletal Interactions

Bhalla-Gehi

Peñuela

Churko

et al. 2010

Journal of Biological Chemistry

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107

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Pannexins (Panx) are a class of integral membrane proteins that have been proposed to exhibit characteristics similar to those of connexin family members. In this study, we utilized Cx43-positive BICR-M1R k cells to stably express Panx1, Panx3, or Panx1-green fluorescent protein (GFP) to assess their trafficking, cell surface dynamics, and interplay with the cytoskeletal network. Expression of a Sar1 dominant negative mutant revealed that endoplasmic reticulum to Golgi transport of Panx1 and Panx3 was mediated via COPII-dependent vesicles. Distinct from Cx43-GFP, fluorescence recovery after photobleaching studies revealed that both Panx1-GFP and Panx3-GFP remained highly mobile at the cell surface. Unlike Cx43, Panx1-GFP exhibited no detectable interrelationship with microtubules. Conversely, cytochalasin B-induced disruption of microfilaments caused a severe loss of cell surface Panx1-GFP, a reduction in the recoverable fraction of Panx1-GFP that remained at the cell surface, and a decrease in Panx1-GFP vesicular transport. Furthermore, co-immunoprecipitation and cosedimentation assays revealed actin as a novel binding partner of Panx1. Collectively, we conclude that although Panx1 and Panx3 share a common endoplasmic reticulum to Golgi secretory pathway to Cx43, their ultimate cell surface residency appears to be independent of cell contacts and the need for intact microtubules. Importantly, Panx1 has an interaction with actin microfilaments that regulates its cell surface localization and mobility.The pannexin family is a new class of integral membrane glycoproteins that have been identified to share sequence homology with the invertebrate gap junction proteins, innexins (1). Unlike the connexin family that is composed of 21 members (2), both the human and mouse genomes contain only three pannexin-encoding genes (Panx1, Panx2, and Panx3) (1). Although connexins and pannexins exhibit no sequence homology, pannexins are predicted to share similar topology with connexins, which includes four transmembrane domains, two extracellular loops, a cytoplasmic loop, and intracellular amino and carboxyl termini (3-5). Our previous study has shown that ectopically expressed Panx1 and Panx3 are capable of trafficking to normal rat kidney (NRK) 2 cell surfaces; however, their distribution profile at cell-cell interfaces is not typically clustered or punctate as seen for Cx43 (5). Consistently, electron micrographs of Panx1 overexpressing Madin-Darby canine kidney cells also revealed dispersed Panx1 localization at the plasma membrane with no evidence of gap junction plaques (4).Cx43 has a relatively short half-life of ϳ1-3 h (6). As a result, Cx43 subunits assembled into connexons within the transGolgi network (7) are constantly being transported to and removed from the plasma membrane (8). Recent reports provide evidence that although Panx1 appears to form similar hexameric channel units defined as pannexons (4), they exhibit slower turnover dynamics in comparison with Cx43, as assessed by the use of pharmacological blocke...

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Retroviral Delivery of Connexin Genes to Human Breast Tumor Cells Inhibits in Vivo Tumor Growth by a Mechanism That Is Independent of Significant Gap Junctional Intercellular Communication

Abstract: The mechanism by which gap junction proteins, connexins, act as potent tumor suppressors remains poorly understood. In this study human breast tumor cells were found to exhibit diverse gap junction phenotypes including (a) undetectable Cx43 and no intercellular communication (

Cited by 199 publications

References 39 publications

Functional Characterization of aGJA1Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma

Functional Characterization of aGJA1Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma

The Gap Junction-independent Tumor-suppressing Effect of Connexin 43

Pannexin1 and Pannexin3 Delivery, Cell Surface Dynamics, and Cytoskeletal Interactions

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