Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca^(2+)-dependent gap junction intercellular communication

Background: Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. Methods: Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca^(2+)-related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. Results: Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca ^(2+ )is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. Conclusions: Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells

Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elu- cidate the reason why the so-called 'bystander effect' mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis. mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by re- verse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malig- nant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. As- sessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was ab- normally located and markedly diminished as compared with normal prostatic epithelial ones, dis- playing a negative correlation to the pathological grade (χ2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indi- cated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein par- ticipated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of 'bystander effect', but also to initiation and progression of prostatic neoplasm.

STUDIES ON THE GAP JUNCTIONAL INTERCELLULARCOMMUNICATION OF HUMAN NASOPHARYNGEALCARCINOMA CELLS AND THE EFFECT OF RII

Human nasopharyngeal carcinoma(NPC) cell line,CNE-2Z, and its clones(L2, H2, L4) with various invasive and metastatic potentials were examined for their gap junctions(GJ), gap junctional intercellular communication(GJIC) and the concentration of cytosolic free calcium(Ca2+). Only a few intermediate junction(IJ)but no GJ structures were observed under electron microscope(EM). CNE-2Z cells showed marked JGIC,while its variants lacked this function using the scraploading dye-transfer technique(SLDT). There was lower concentration of[Ca2+]. in L2 cells(a variant with high invasive and metastatic Potential) compared to that in H2 and L4 cells(variants with medium and low invasive and metastatic Potentials, respectively). These data suggested that high invasive and metastatic potentials might be correlated with the levcl of[Ca2+]i in NPC cells.The effect of RII(4-hydroxycarbophenyl retinamide) on NPC cells also investigated, After 3-7 d of RII(10-5 M) treatment, there was no change in the number of gap junctions and other kind of intercellular junctions in NPC cells observed under EM. The JGIC of CNE-2Z weaked and then disappeared finally with prolonging of RII treatment. However. there was no influence on its variants. The level of[Ca2+], in NPC cells apparently fell after 6 h of RII treatment, and rose to original level with persisting of RII treatment. Whether the fluctuating of[Ca2+]i level is related to the inhibitory effect of RII treatment on growth and invasion of NPC cells needs to be further studied.

Basic Investigations EXPRESSION OF GAP JUNCTION PROTEIN Cx43 IN CULTURED HUMAN NORMAL AND MALIGNANT LUNG CELLS

Gap junctional intercellular communicationexchange of small molecules and ions between contiguous cells through membranous gap junctional channelsis essential for growth control and tissue homecotasis. This work concerns the functional expression of gap junction protein connexin 43 (Cx43) in normal human lung cells and the changes in lung carcinoma cells. By. using Northern blot hybridization analysis and Cx43 immunocytochemical methods, it was otherved that cultured normal human embryonic lung cells expressed a high level of Cx43 in both mRNA and protein levels.The Cx43 immunofluorescence was localized at cell membrane regions corresponding to the location of gap junctions. These normal lung cells were competent of intercellular communication function as detected by Lucifer yellow dye transfer. In contrast to normal celis, Cx43 mRNA and protein was not detectable in the carcinoma PG cell line. These tumor cells were defective of intercellular communication function. These results demonstrate that Cx43 is expressed in normal cultured human embryonic lung cells but not in lung tumor cells. The lack of intercellular communication in the lung tumor cell line correlates with dysfunctional intercellular communication. The suggestive role of Cx as a tumor suppersor gene is discussed.

Genotoxic and Nongenotoxic Effects of Glycidyl Methacrylate on Human Lung Fibroblast Cells

Objective To evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro. Methods DNA strand breakage was determined by single cell gel electrophoresis, and DNA ladder formation assay and flow cytometric analysis were carried out to detect apoptic responses of cells to GMA exposure. The HPRT gene mutation assay was used to evaluate the mutagenicity, and the effect of GMA on gap junctional intercellular communication (GJIC) in the exposed cells was examined with the scrape loading/dye transfer technique. The ability of GMA to transform 2BS cells was also tested by an in vitro cell transformation assay. Results Exposure to GMA resulted in a dose-dependent increase in DNA strand breaks but not apoptic responses. GMA was also shown to significantly induce HPRT gene mutations and morphological transformation in 2BS cells in vitro. In contrast, GMA produced a concentration-dependent inhibition of GJIC. Conclusions GMA elicits both genotoxic and nongenotoxic effects on 2BS cells in vitro. The induction of DNA damage and gene mutations and inhibition of GJIC by GMA may casually contribute to GMA-induced cell transformation.

Connexin 40-formed GJIC increases the phototoxicity of photodynamic therapy through ROS-and calcium-mediated pathways

OBJECTIVE To explore the effect of connexin(Cx)40-formed gap junctional intercellular communication(GJIC)on Photofrin-photodynamic therapy(PDT)phototoxicity in Cx40-transfected He La cells and its potential mechanisms.METHODS He La cell line stably transfected to express Cx40 was seeded at high and low cell density,respectively,to assess in vitro photosensitivity using CCK8 assay.Western blot assay was performed to detect the expression of Cx40.The intracellular ROS and Ca^(2+) concentrations were determined using flow cytometer.4-HNE and ceramide were measured using ELISA assay.RESULTS Cx40-composed GJ formation at high density enhances the phototoxicity of PhotofrinPDT.When the Cx40 is not expressed or Cx40 channels are blocked,the phototoxicity in high-density cultures substantially reduces,indicating that the enhanced PDT phototoxicity at high density is mediated by Cx40-composed GJIC.The GJIC-mediated increase in PDT phototoxicity was associated with ROS and calcium-mediated stress signaling pathways.CONCLUSION The work uniquely presents the ability of Cx40-composed GJIC to enhance the sensitivity of malignant cells to PDT,and indicates that maintenance or increase of Cx40-formed GJIC may be a profitable strategy towards the enhancement of PDT therapeutic efficiency.