Increased gap junctional intercellular communication capacity and connexin 43 and 26 expression in rat bladder carcinogenesis

Many reports have suggested that gap junctional intercellularcommunication or gap junction proteins (connexins) could havetumor suppression characteristics. We investigated gap junctionalintercellular communication capacity and connexin 26, 32 and43 mRNA expression in four rat bladder cell lines and the resultswere compared to their tumorigenicity. We also examined connexinexpression in rat bladder carcinomas induced by 3,2'-dlmethyl-4-aminobiphenylor N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) and in normalbladders. There was clear tendency that cell lines with greatercommunication had stronger tumorigenicity and more expressionof connexin 26 or 43. We could not detect connexin 32 in thesecell lines. In normal bladder tissue, connexin 43 expressionwas barely detectable and there was no detectable connexin 26.However, in rat bladder carcinomas, especially the EHBNinducedcarcinomas, abundant expression of both connexins was observed.These results indicate that increased gap junctional intercellularcommunication capacity or increased connexin(s) expression maygive a growth advantage in rat bladder carcinogenesis.     

Upregulation of connexin 43 protein expression and increased gap junctional communication by water soluble disodium disuccinate astaxanthin derivatives

Carotenoids are plant pigments whose consumption is associated with lower cancer rates in humans. Studies in experimental animal and cell systems have confirmed the cancer chemopreventive activity of these compounds. However, their extremely hydrophobic nature makes these compounds biologically unavailable unless delivered in organic solution to model systems. We have synthesized novel disodium salt disuccinate astaxanthin derivatives that possess high aqueous dispersibility. When delivered to mouse embryonic fibroblast C3H/10T1/2 cell cultures, either in aqueous or aqueous/ethanol solutions, these derivatives are biologically active. Biological activity was demonstrated by (1) upregulated expression of connexin 43 (Cx43) protein; (2) increased formation of Cx43 immunoreactive plaques in regions of the plasma membrane consistent with localization of gap junctions; (3) significantly upregulated gap junctional intercellular communication (GJIC) as demonstrated by Lucifer Yellow dye transfer after microinjection (P < 0.03; Fisher's Exact test). Enhanced expression of Cx43 and increased GJIC have been previously demonstrated to result in inhibition of in vitro neoplastic transformation of 10T1/2 cells as well as growth reduction of human tumors in xenografts. These novel derivatives possess increased utility as water soluble and water dispersible agents, allowing for aqueous delivery both in vitro and in vivo, properties that could enhance their potential clinical utility as potent cancer chemopreventive agents.     

Disruption of gap junctional intercellular communication by lindane is associated with aberrant localization of connexin43 and zonula occludens-1 in 42GPA9 Sertoli cells

Lindane (gamma-hexachlorocyclohexane) is a lipid-soluble pesticide that exerts carcinogenic and reprotoxic properties. The mechanisms by which lindane alters testicular function are unclear. Sertoli cells control germ cell proliferation and differentiation through cell-cell communication, including gap junction intercellular communication. Using the 42GPA9 Sertoli cell line, we show that lindane, at a non-cytotoxic dose (50 microM), abolished gap junction intercellular communication (GJIC) between adjacent cells. This change was associated with a time-related diminution and redistribution of Cx43 from the membrane to the cytoplasmic perinuclear region. A similar alteration was observed for ZO-1, a tight junction component associated with Cx43, but not for occludin, an integral tight junction protein. After a 24 h lindane exposure, Cx43 and ZO-1 colocalized within the cytoplasm and no modification of non-phosphorylated and phosphorylated isoforms of Cx43 was observed. By double immunofluorescent labelling we demonstrate that the cytoplasmic Cx43 signal was not present in either the endoplasmic reticulum/Golgi apparatus or lysosomes. These results suggest that lindane inhibits GJIC between Sertoli cells and that aberrant Cx43/ZO-1 localization may be responsible for this effect. The alterations in gap junctions induced by lindane in 42GPA9 Sertoli cells are similar to those observed in tumour cells and may be involved in the pathogenesis of neoplastic seminomal proliferation.     

Suberoylanilide hydroxamic acid enhances gap junctional intercellular communication via acetylation of histone containing connexin 43 gene locus

A histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induces apoptosis in neoplastic cells, but its effect on gap junctional intercellular communication in relation to apoptosis was unclear. Therefore, we carried out a comparative study of the effects of two HDAC inhibitors, SAHA and trichostatin-A, on gap junctional intercellular communication in nonmalignant human peritoneal mesothelial cells (HPMC) and tumorigenic ras oncogene-transformed rat liver epithelial cells (WB-ras) that showed a significantly lower level of gap junctional intercellular communication than did HPMC. Gap junctional intercellular communication was assessed by recovery rate of fluorescence recovery after photobleaching. Treatment of HPMC with SAHA at nanomolar concentrations caused a dose-dependent increase of recovery rate without inducing apoptosis. This effect was accompanied by enhanced connexin 43 (Cx43) mRNA and protein expression and increased presence of Cx43 protein on cell membrane. Trichostatin-A induced apoptosis in HPMC but was less potent than SAHA in enhancing the recovery rate. In contrast, treatment of WB-ras cells with SAHA or trichostatin-A induced apoptosis at low concentrations, in spite of smaller increases in recovery rate, Cx43 mRNA, and protein than in HPMC. Chromatin immunoprecipitation analysis revealed that SAHA enhanced acetylated histones H3 and H4 in the chromatin fragments associated with Cx43 gene in HPMC. These results indicate that SAHA at low concentrations selectively up-regulates Cx43 expression in normal human cells without induction of apoptosis, as a result of histone acetylation in selective chromatin fragments, in contrast to the apoptotic effect observed in tumorigenic WB-ras cells. These results support a cancer therapeutic and preventive role for specific HDAC inhibitors.     

Upregulation of gap junctional communication and connexin43 gene expression by carotenoids in human dermal fibroblasts but not in human keratinocytes

Consumption of dietary carotenoids has been statistically associated with decreased risk of cancer at several anatomic sites. In a model murine system of carcinogenesis (the 10T1/2 assay), we have previously shown that carotenoids can inhibit chemically and physically induced neoplastic transformation. This action is strongly correlated with the ability of carotenoids to increase gap-junctional communication (GJC) by induction of connexin43 (Cx43) gene expression. Here we extend these studies to human foreskin-derived dermal fibroblasts and keratinocytes. In fibroblasts, β-carotene and canthaxanthin at concentrations between 10^?5 and 3 × 10^?6 M were found to strongly enhance GJC in a dose- and time-dependent manner. This was accompanied by an increase in the number of immunofluorescent junctional plaques recognized by an anti-Cx43 antibody and by an increase in Cx43 protein level as determined by western blot analysis. No decrease in proliferation rates was detected by [H³]thymidine labeling. Human keratinocytes grown in monolayer culture did not respond to carotenoids in terms of GJC as measured by dye transfer, immunofluorescent analysis of Cx43 distribution, or Cx43 levels as measured by western blotting. Both cell types accumulated high levels of carotenoids. Because canthaxanthin, which has no known provitamin A activity in mammals, is as active in fibroblasts as is β-carotene, the carotenoid with the highest provitamin A activity, the induction of GJC and Cx43 expression by carotenoids in human dermal fibroblasts seems unrelated to their provitamin A status. The lack of response of keratinocytes suggests differences in regulation of Cx43 expression or in carotenoid processing. © 1995 Wiley-Liss Inc.     

Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells

Cell to cell communication via gap junctions is essential in the maintenance of the homeostatic balance of multicellular organisms. Aberrant intercellular gap junctional communication (GJIC) has been implicated in tumor promotion, neuropathy and teratogenesis. Oxidative stress has also been implicated in similar pathologies such as cancer. We report a potential link between oxidative stress and GJIC. Hydrogen peroxide, a known tumor promoter, inhibited GJIC in WB-F344 rat liver epithelial cells with an I50 value of 200 microM. Inhibition of GJIC by H2O2 was reversible as indicated by the complete recovery of GJIC with the removal of H2O2 via a change of fresh media. Free radical scavengers, such as t-butyl alcohol, propylgallate, and Trolox, did not prevent the inhibition of GJIC by H2O2, which indicated that the effects of H2O2 on GJIC was probably not a consequence of aqueous free radical damage. The depletion of intracellular GSH reversed the inhibitory effect of H2O2 on GJIC. The treatment of glutathione- sufficient cells with H2O2 resulted in the hyperphosphorylation of connexin43, which is the basic subunit of the hexameric gap junction protein, as determined by Western blot analysis. TPA, a well-known tumor promoter, also inhibits GJIC via hyperphosphorylation of GJIC, which is a result of protein kinase-C activation. However, H2O2 also induced hyperphosphorylation in GSH-deficient cells that had normal rates of GJIC. Therefore, the mechanism of GJIC inhibition must be different from the TPA-pathway and involves GSH.      

Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C

In this study, we investigated whether the tumor promoters, 12-O- tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), and 1,1- bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), inhibited gap junctional intercellular communication (GJIC) in a cell-specific or connexin-specific manner and whether protein kinase C was involved. To do this, we used highly communicating WB-F344 rat liver epithelial cells, which express connexin43 as their predominant gap junction protein, WB-aB1 cells, which are a GJIC-incompetent mutant line of WB- F344 cells and that express connexin43, WB-a/32-10 cells, which are a highly communicating derivative of WB-aB1 cells generated by stable transduction with a connexin32 retroviral expression vector, and primary cultured rat hepatocytes, which express conexin32 predominantly. Treatment of WB-F344 and WB-a/32-10 cells, but not hepatocytes, with TPA inhibited GJIC (assayed by Lucifer Yellow dye microinjection). This inhibition involved protein kinase C because (i) inhibition was prevented by co-treatment of the cells with a specific protein kinase C inhibitor, bis-indolylmaleimide, and (ii) treatment with TPA for 24 h had no effect on dye-coupling in agreement with the downregulation of protein kinase C. TPA also caused the internalization of Cx43-containing gap junctions and the formation of a hyperphosphorylated form of Cx43, Cx43-P3, in WB-F344 cells only, but TPA had no effect on Cx32-containing gap junctions or protein mobility. In contrast, PB inhibited GJIC only in hepatocytes and DDT inhibited GJIC in all three types of cells; bis-indolylmaleimide did not block the effects of either agent. These results indicate that the inhibitory actions of TPA and PB on GJIC are cell-specific rather than connexin- specific and that TPA inhibits connexin43 and connexin32-mediated GJIC through a protein kinase C-dependent mechanism.      

Inhibition of gap junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hyperphosphorylated

Polyunsaturated fatty acids have attracted much interest dueto their wide spectrum of biological activities which includethe modulation of gap junctional communication (GJC). Sincegap junctions play critical roles in maintaining the functionalintegrity of organs and tissues, and loss of intercellular communicationis associated with a number of pathological conditions, we investigatedthe effects of the n - 6 and n - 3 series of polyunsaturatedfatty acids and their derivatives on GJC in WB cells as determinedby the ability of Lucifer Yellow-loaded cells to transfer thedye to neighbouring recipient cells. Studies were also conductedto investigate the possible mechanisms of action of the fattyacids. Treatment of cells with 10 µM arachidonic acid(20: 4 n - 6) resulted in a rapid and transient loss of communicationcompetence. The response to 20µM 20: 4 (n - 6) was prolonged(>210 min) but was readily reversible by washing the cellswith fatty acid-free bovine serum albumin. Cells which had regainedtheir communication competence responded to further additionsof 20: 4 (n - 6). The fatty acids, 18: 3 (n - 6), 20: 5 (n -3), 22: 6 (n - 3) and the 15-hydroxy- and the 15-hydroperoxy-derivativesof 20: 4 (n - 6) were also powerful inhibitors of GJC, while23: 4 (n - 6) was a relatively weak inhibitor. The saturated20 carbon fatty acid, 20: 0, and the methyl ester of 20: 4 (n- 6) were without effect. This illustrates the importance ofunsaturation and the carboxyl group as structural requirementsfor activity. 20: 4 (n - 6)-induced inhibition of dye transferwas not attenuated by pretreating the cells with either phorbol-12-myristate-13-acetate(PMA) or indomethacin, suggesting that regulation of gap junctionalpermeability by 20: 4 (n - 6) in WB cells was neither dependenton PMA-responsive isozymes of protein kinase C nor requiredthe metabolism of the fatty acids by cyclo-oxygenase. However,the effect of 20: 4 (n - 6) was antagonized by preincubatingWB cells with either nordihydroguai-aretic acid or (±)-isoproterenoland isobutylmethyl-xanthine. Western blot analysis of connexin43 (Cx43), the major gap junctional protein expressed in thesecells, revealed no detectable changes to the electrophoreticmobility of Cx43 even after 60 min of incubation in the presenceof 20: 4 (n - 6). As expected, other inhibitors of gap Junetionalpermeability including epidermal growth factor, phorbol esteror lysophosphatidic acid induced a retardation in the mobilityof Cx43, indicating an enhancment in the phosphorylation ofCx43 protein. The data indicate that inhibition of GJC by 20:4 (n - 6) has a novel mechanism which does not require phosphorylationof Cx43 but may require its metabolism to eicosanoids. In additionthe inhibitory effect of 20: 4 (n - 6) can be modulated by anincrease in intracellular cAMP concentration which has beenreported to enhance cell-cell communication. The data also argueagainst a non-specific detergent action of the fatty acids.     

Inhibition of gap junctional intercellular communication and enhancement of growth in BALB/c 3t3 cells treated with connexin43 antisense oligonucleotides

Many studies have correlated reductions in gap junctional intercellular communication (a) with altered cellular growth, tumor promotion, and neoplastic transformation. To test directly whether reduced GJIC affects cellular growth, GJIC was inhibited in murine BALB/c 3T3 fibroblasts by treatment with a phosphorothioatemodified antisense oligonucleotide targeted against the connexin43 translation start codon, and in vitro cell growth was monitored. The cells were incubated with the oligonucleotide (0.1-0.5 μM) in liposomes in serumless culture medium for 16 h; washed and refed with serum-containing medium; and analyzed for dye-coupling, connexin43 protein and mRNA levels, and cell growth over the next 5 d. The antisense oligonucleotide inhibited dye-coupling and reduced connexin43 protein levels in a concentration-dependent manner but had no effect on connexin43 mRNA levels. Cell growth rate was not affected, but saturation density was increased approximately threefold by the oligonucleotide. These data support a role for GJIC in the establishment of contact inhibition of in vitro cell growth. © 1995 Wiley- Liss, Inc.     

Exogenous CXCL12 activates protein kinase C to phosphorylate connexin 43 for gap junctional intercellular communication among confluent breast cancer cells

Despite ongoing attempts to improve the overall breast cancer (BC) survival rate, BC cells’ (BCCs) predilection for metastasizing to the bone marrow has enabled BCCs to not only remain dormant, but also evade detection. BCCs are able to acquire quiescence by establishing gap junctional intercellular communication (GJIC) with the stroma through the assembly of connexins (Cxs). The chemoattractant CXCL12 also appears to play a role in GJIC based on its tendency to decrease when GJIC is formed between BCCs and bone marrow stroma. This study investigates the role CXCL12 has on Cx43 expression and PKC-mediated Cx43 phosphorylation. Cx43 gene reporter assays revealed that as the BCCs come in contact with each other and establish GJIC, there is an inverse relationship between CXCL12 level and Cx43 expression. Immunoblot analyses confirmed this relationship at the level of protein, showing decreased Cx43 and reduced Cx43 phosphorylation at higher CXCL12 concentrations. However, real-time PCR studies revealed little change in Cx43 mRNA levels, despite stimulation with different concentrations of CXCL12, indicating CXCL12’s effect on Cx43 is post-translational, through phosphorylation. Immunoblot analyses and functional dye exchange studies showed activation of PKC by exogenous CXCL12 in the phosphorylation, which in turn, increased intercellular communication. These findings elucidate the importance of considering the microenvironment’s role in micrometastasis in clinical studies pertaining to prospective breast cancer treatment.     

Gap junctional intercellular communication and connexin43 expression in human ovarian surface epithelial cells and ovarian carcinomas in vivo and in vitro.

Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) are frequently decreased in neoplastic cells and have been increased by cAMP and retinoids. GJIC and connexin expression were investigated in early passage normal human ovarian surface epithelial (HOSE) cells, human ovarian adenocarcinoma cell lines (CaOV-3, NIH:OVCAR-3, SK-OV-3 and SW626) and surgical specimens of human serous cystadenocarcinomas. We hypothesized that GJIC and connexin expression would be decreased in neoplastic cells and would be increased by cAMP and retinoic acid. Cultured HOSE cells exhibited extensive fluorescent dye-coupling and connexin43 (Cx43) expression; other connexins were not detected. The ovarian adenocarcinoma cell lines had little dye-coupling or connexin expression. Deletions and rearrangements of the Cx43 gene were not detected by Southern blotting in the carcinoma lines. N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate and all-trans-retinoic acid inhibited cell proliferation, but did not enhance GJIC or Cx43 expression. Surface epithelial cells of benign ovaries expressed Cx43, but this expression was barely detectable in ovarian serous cystadenocarcinomas. Thus, normal HOSE cells had extensive GJIC and Cx43 expression whereas ovarian carcinoma cells had less and cAMP and retinoic acid did not change these, although both agents inhibited cell growth.     

Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant-negative connexin 43

Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB-F344 rat liver epithelial or mouse osteoblastic MC3T3-E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY-457) or LY with a MW of 649 Da (LY-649). Transfection of a dominant negative-connexin 43 (Cx43) gene decreased GJIC (>50%) when LY-649 was used, however, normal GJIC was observed in both cell lines when LY-457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3-E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3-E1. WB-F344 cells normally exhibit a biphasic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network-like structures by WB-F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild-type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals.     

Modulation of gap junctional intercellular communication by EGF in human kidney epithelial cells

Modulation of gap junctional intercellular communication (GJIC)was studied in a multistep model of human renal epithelial carcinogenesis.We report that the majority of primary human kidney epithelialcells (NHKE) grown from fetal kidney explants did not communicatethrough gap junctions. Communication could, however, be observedwithin a subpopulation of the cells. Ni(II)-immortalized cells(IHKE) showed GJIC at a level of 10–20 communicating cells,but with heterogeneous regions on the dish, with regard to bothcommunication and distribution of connexin43. The heterogeneitywas less pronounced in a ras-transfected tumourigenic cell line(THKE), which also showed communication of     

Altered gap junctional intercellular communication in neoplastic rat esophageal epithelial cells

Gap junctional intercellular communication (GJIC) is reduced in many neoplastic cells, but few data exist for esophageal neoplasms. GJIC was examined by fluorescent dye microinjection in two nontumorigenic and two highly tumorigenic rat esophageal epithelial cell lines. All lines expressed high levels of dye coupling in homologous cell culture. In cocultures of nontumorigenic and tumorigenic cells, however, only one of six cell combinations displayed significant heterologous GJIC. Northern, Western, and immunohistochemical analyses indicated that all four cell lines expressed comparable levels of connexin43 (Cx43), but not connexin32 or connexin26, and formed Cx43-containing gap junction plaques at cell-cell interfaces. Immunostaining of rat esophageal frozen sections demonstrated that esophageal epithelial cells expressed Cx43 in vivo. In normal epithelium, the highest expression was seen in the basal cells and little suprabasal staining was evident. In preneoplastic and neoplastic lesions of the esophageal epithelium which were induced by treating rats with N-nitrosomethylbenzylamine, Cx43 staining of the basal layer was also seen but appeared to be more diffuse compared to normal epithelium. In addition, suprabasal Cx43 staining was apparent in dysplastic and papillomatous lesions. These results indicate that Cx43 is expressed in normal and neoplastic rat esophageal cells and that the cells exhibit extensive homologous GJIC, but little heterologous GJIC. This lack of heterologous GJIC may be due to differences in cell adhesion proteins or other factors.      

Increased gap junctional intercellular communication in Syrian hamster embryo cells treated with oxidative agents

The effects of K₂CrO₄, H₂O₂, benzoyl peroxide, menadione, KBrO₃and UV_365nm on gap junctional intercellular communication (GJIC)have been studied in the 12-O-tetra-decanoylphorbol-13-acetate(TPA)-sensitive Syrian hamster embryo (SHE) cell line BPNi.All agents were found to increase the level of GJIC by 50–100%.Also, in early passage SHE cells, a tendency for increased GJICwas found for the oxidative agents studied. Hydrogen peroxidewas used as a model compound in the subsequent studies. Theincrease in GJIC was reversible, and it was not due to an increasednon-junctional permeability. Hydrogen peroxide counteractedthe TPA-induced decrease in GJIC, regardless of whether thecells were exposed to the compounds simultaneously or the cellswere pre-exposed to TPA before addition of H₂O₂. The GJIC enhancementby H₂O₂ was slightly reduced by the addition of the hydroxylradical scavenger dimethylsulphoxide or by the inhibition ofcatalase by amitrole. The cAMP/ protein kinase A system is theonly characterized signal transduction system that is knownto increase GJIC in most cell types. Hydrogen peroxide did notincrease the amount of cAMP (or cGMP) in BPNi cells, while forskolinand a phosphodiesterase inhibitor had to increase the cAMP levelseveral-fold to affect GJIC to the same degree as the oxidativeagents. Some inhibitors of protein kinase A were assayed fortheir ability to inhibit the increases in GJIC caused by H₂O₂and forskolin. Staurosporine inhibited the forskolin-inducedincrease in GJIC, with much less effect on the H₂O₂-inducedincrease. H8, H88 and H89 had less effect than staurosporineon the forskolin-induced increase in GJIC. The results suggestthat the cAMP/protein kinase A system may not be involved inthe increase in GJIC caused by H₂O₂, although this cannot becompletely ruled out.     

Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells

Two flavones, apigenin and tangeretin, were studied for theirability to modulate gap junctional intercellular communication(GJIC) in the rat liver epithelial cell line REL. Their cytotoxicitywas first determined by cell density and neutral red uptakeassays: neither apigenin nor tangeretin are cytotoxic at 10and 25 µM, the concentrations used in our experiments.We then studied GJIC using the dye transfer assay and we observedthat both apigenin and tangeretin enhance it, the maximum stimulation(x 1.7–1.8) being achieved at 25 µM for 24 h. Whenthe dye transfer was enhanced, the amount of connexin 43 increased,which was demonstrated by Western blot and immunofluorescenceanalysis. For apigenin only, Northern blot analysis showed anaccumulation of connexin 43 mRNA. In addition, the incubationof REL cells with the two compounds, for 1 or 24 h, preventedthe inhibition of dye transfer by 12-O-tetradecanoylphorbol-13-acetate(1or 10 ng/ml). The enhancement of GJIC by apigenin could be oneof the major mechanisms responsible for apigenin's anti-tumourpromoting action in vivo. As for tangeretin, its capacity toenhance GJIC completes its potential protective properties towardsthe post-initiation process.