Norbornyne: a cycloalkyne reacting like a dicarbene.
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Letter↗
Biomedical subjects
D W Laird
Publications and source records attributed to D W Laird.
DFT study of the cycloaddition reactions of strained alkynes.
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Letter↗
Comparative analysis and application of fluorescent protein-tagged connexins.
In order to examine connexin transport, assembly, and turnover in living cells, we tagged green fluorescent protein or its color variants to several members of the connexin family of proteins. When green fluorescent protein was tagged to the carboxyl terminal end of connexin43 (Cx43-GFP), the resulting fusion protein was transported and assembled into functional gap junctions. However, when GFP was tagged to the amino terminal end of Cx43 (GFP-Cx43), this chimera was biosynthesized, transported to the plasma membrane, but failed to form gap junction channels that could transfer Lucifer yellow. Single cells that expressed Cx43-GFP were capable of transporting this fusion protein to the cell surface in the absence of cell-cell contact. Imaging of Cx43-yellow (Y)FP (Cx43-YFP) was quite efficient; however, the low quantum yield Cx43-BFP and the requirement for ultraviolet excitation made this chimera less suitable for time-lapse imaging. Cx43-cyan C(FP) (Cx43-CFP) was more suitable for imaging than Cx43-blue (B)FP and could be effectively separated from Cx43-YFP. The versatility of tagging GFP to the carboxyl terminal end of other members of the connexin family was established when Cx32-GFP and Cx26-YFP were found to assemble into gap junctions capable of transferring Lucifer yellow. Finally, we are examining the effectiveness of using a new red fluorescent protein (DsRed) fused to connexins in combination with Cx-GFP to simultaneously examine the kinetics, transport and turnover of two connexins. Together, our studies suggest that tagging fluorescent proteins to the carboxyl terminal end of connexins is an effective and valuable approach for studying the life cycle and dynamics of connexins in living cells.
Animals↗
The origin of annular junctions: a mechanism of gap junction internalization.
Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3-6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.
Animals↗
Expression and imaging of connexin-GFP chimeras in live mammalian cells.
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Animals↗
Regulation of the gap junction connexin 43 gene by androgens in the prostate.
Androgens play an important role in prostate gland development and function, and have been implicated in prostate carcinogenesis. We report the regulation of the gap junctional intercellular communication gene connexin 43 (Cx43) by androgens in the prostate gland. In rat ventral prostate tissue, only trace levels of Cx43 mRNA were detected. Castration, however, resulted in a high increase in Cx43 mRNA and protein. Cx32 was unchanged. Castration-induced Cx43 mRNA and protein were abolished by administration of dihydrotestosterone (DHT). Following castration, prostate weights were approximately 16% of sham-treated controls. However, DHT replacement resulted in prostate weights which were not different from sham-treated controls. Under similar castration conditions, Cx43 induction coincided with pronounced apoptosis in the prostate gland cells, and DHT prevented the induction of apoptosis. Given the physiological role of gap junctions and androgens in the regulation of prostate tissue homeostasis, our observations are relevant to the understanding of androgen-dependent prostate carcinogenesis.
Animals↗
Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions.
Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gap junctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.
Actin Cytoskeleton↗
Aggregated DsRed-tagged Cx43 and over-expressed Cx43 are targeted to lysosomes in human breast cancer cells.
To investigate if either wild-type or aggregated Cx43 is abnormally targeted to lysosomes in human breast tumor cells, we examined the fate of DsRed-tagged Cx43 and over-expressed Cx43 in communication-deficient HBL-100 and MDA-MB-231 cells. DsRed-tagged Cx43 was assembled into gap junctions in control normal rat kidney cells that express endogenous Cx43 but not in Cx43-negative HBL-100 cells. However, when HBL-100 cells were engineered to coexpress wild-type Cx43 a population of DsRed-tagged Cx43 was rescued and assembled into gap junctions. Co-expression of wild-type Cx26 failed to rescue the assembly of DsRed-tagged Cx43 into gap junctions. Immunolocalization studies revealed that DsRed-tagged Cx43 was aggregated and partially localized to lysosomes. Interestingly, when human MDA-MB-231 breast tumor cells over-expressed wild-type Cx43, Cx43 protein primarily localized to lysosomes. Together, these studies provide evidence for Cx43 being targeted to lysosomes as a result of misfolding and aggregation, while in other cases, the delivery of wild-type Cx43 to lysosomes appears to be due to defects innate to the breast tumor cell type.
Animals↗
Neuronal differentiation and growth control of neuro-2a cells after retroviral gene delivery of connexin43.
Given the roles proposed for gap junctional intercellular communication in neuronal differentiation and growth control, we examined the effects of connexin43 (Cx43) expression in a neuroblastoma cell line. A vesicular stomatitis virus G protein (VSVG)-pseudotyped retrovector was engineered to co-express the green fluorescent protein (GFP) and Cx43 in the communication-deficient neuro-2a (N2a) cell line. The 293 GPG packaging cell line was used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or chimeric Cx43.GFP fusion protein. The titer of viral supernatant, as measured by flow cytometry for GFP fluorescence, was approximately 2.0 x 10(7) colony form units (CFU)/ml and was free of replication-competent retroviruses. After a 7-day treatment with retinoic acid (20 microm), N2a transformants (N2a-Cx43 and N2a-Cx43.GFP) maintained the expression of Cx43 and Cx43.GFP. Expression of both constructs resulted in functional coupling, as evidenced by electrophysiological and dye-injection analysis. Suppression of cell growth correlated with expression of both Cx43 or Cx43.GFP and retinoic acid treatment. Based on morphology and immunocytochemistry for neurofilament, no difference was observed in the differentiation of N2a cells compared with cells expressing Cx43 constructs. In conclusion, constitutive expression of Cx43 in N2a cells does not alter retinoic acid-induced neuronal differentiation but does enhance growth inhibition.
Cell Differentiation↗
Regulation of fas ligand expression during activation-induced cell death in T cells by p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.
Activation-induced cell death (AICD) is a mechanism of peripheral T cell tolerance that depends upon an interaction between Fas and Fas ligand (FasL). Although c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) may be involved in apoptosis in various cell types, the mode of regulation of FasL expression during AICD in T cells by these two MAPKs is incompletely understood. To investigate the regulatory roles of these two MAPKs, we analyzed the kinetics of TCR-induced p38 MAPK and JNK activity and their regulation of FasL expression and AICD. We report that both JNK and p38 MAPK regulate AICD in T cells. Our data suggest a novel model of T cell AICD in which p38 MAPK acts early to initiate FasL expression and the Fas-mediated activation of caspases. Subsequently, caspases stimulate JNK to further upregulate FasL expression. Thus, p38 MAPK and downstream JNK converge to regulate FasL expression at different times after T cell receptor stimulation to elicit maximum AICD.
Animals↗
Clustering of connexin 43-enhanced green fluorescent protein gap junction channels and functional coupling in living cells.
Communication-incompetent cell lines were transfected with connexin (Cx) 43 fused with enhanced green fluorescent protein (EGFP) to examine the relation between Cx distribution determined by fluorescence microscopy and electrical coupling measured at single-channel resolution in living cell pairs. Cx43-EGFP channel properties were like those of wild-type Cx43 except for reduced sensitivity to transjunctional voltage. Cx43-EGFP clustered into plaques at locations of cell-cell contact. Coupling was always absent in the absence of plaques and even in the presence of small plaques. Plaques exceeding several hundred channels always conferred coupling, but only a small fraction of channels were functional. These data indicate that clustering may be a requirement for opening of gap junction channels.
Animals↗
Role of HSP90 in mediating cross-talk between the estrogen receptor and the Ah receptor signal transduction pathways.
Tetrachlorodibenzo-p-dioxin (TCDD)-mediated gene transactivation via the Ah receptor (AhR) has been shown to be dependent upon estrogen receptor (ER) expression in human breast cancer cells. We have investigated the 90-kDa heat shock protein (HSP90) as a mediator of cross-talk between the AhR and the ER signal transduction pathways. The effect of HSP90 overexpression on receptor activity was determined by transient transfection assays using a HSP90 expression vector. Ligand-inducible gene expression was inhibited when the HSP90 expression vector was cotransfected with a TCDD-responsive reporter plasmid. However, overexpression of HSP90 did not block induction of an estrogen-responsive reporter plasmid. To determine whether ER facilitates AhR signaling through its ability to squelch HSP90, two vectors expressing protein products that bind HSP90 were transfected into MDA-MB-231 cells. Introduction of (i) He11, an ER deletion mutant that does not bind DNA, and (ii) the ligand-binding domain of human AhR, both led to increased basal and TCDD-inducible CYP1A1 expression. Finally, the subcellular distribution of HSP90 was investigated in human breast cancer cell lines. These studies showed HSP90 to be primarily cytoplasmic in ER-positive cell lines, whereas in matched ER-negative cell lines HSP90 was distributed equally between the cytoplasm and nucleus. Taken together, these results demonstrate that HSP90 can regulate AhR activity in vivo, and that Ah-responsiveness is dependent upon cellular ER content through a mechanism that involves HSP90.
Breast Neoplasms↗
Deficiency of connexin43 gap junctions is an independent marker for breast tumors.
Gap junctions are intercellular channels that are formed from members of a family of proteins, the connexins (Cxs). Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Here, we examined the expression of Cx43, a major Cx in breast tissue, in 32 surgical specimens obtained from breast cancer patients who underwent a primary surgical resection prior to chemotherapy or radiotherapy treatments. The expression of Cx43 gap junctions was compared to the levels of estrogen, progesterone, and erbB2 tyrosine kinase receptors. In addition, a panel of breast cancer cell lines and a series of normal rat mammary tissues and rat mammary tumors induced in vivo by dimethylbenz(a)anthracene were studied. We demonstrated that the lack of Cx43 gap junctions is a common feature of human mammary cancer tissues compared to nonneoplastic breast tissues surrounding primary tumors. Cx43 gap junctions were not observed in ductal carcinomas in situ, infiltrating ductal carcinomas, and infiltrating lobular carcinomas, and they seem to be independent of estrogen, progesterone, and erbB2 receptor status. In breast cancer cell lines and rodent mammary carcinoma tissues, down-regulation of Cx43 occurs at the mRNA level, suggesting a transcriptional mechanism for the decrease of Cx43 protein in breast cancer. In summary, this study provides evidence of decreased expression of Cx43 gap junctions in breast cancer at various stages of progression as well as breast cancer cell lines and raises the possibility that Cx43 may be a useful marker for detecting early oncogenesis in the breast. Because Cx43 gap junctions are lacking in breast cancer and restoration of Cx43 has been shown to reverse the malignant phenotype in vitro, pharmacological up-regulation of Cx43 may prove beneficial in cancer therapeutics.
Adult↗
Quiescent mammary epithelial cells have reduced connexin43 but maintain a high level of gap junction intercellular communication.
Gap junctions have been implicated in growth control, but it remains unclear whether cells that enter a quiescent state continue to express connexins and maintain a high level of gap junction intercellular communication (GJIC). To this end, MAC-T cells, a bovine mammary epithelial cell line, were serum starved for 48 h to induce a quiescent (G0) state. In quiescent cells, [3H]thymidine incorporation was reduced by 97.3% from serum-fed controls. Western blotting in conjunction with Phosphorlmager analysis revealed up to a 20-fold decrease in the expression of the gap junction protein connexin43 (Cx43 or alpha 1) and a shift toward the unphosphorylated form in quiescent cells. However, cell-to-cell transfer of the gap junction-permeable fluorescent tracer, Lucifer yellow, was only moderately reduced in quiescent cells. In control cells, Cx43 was predominantly perinuclear, although it was also present at sites of cell-cell apposition. In quiescent cells, intracellular labeling for Cx43 decreased without a corresponding reduction at areas of cell-cell contact. Recovery from serum deprivation resulted in increased thymidine incorporation that corresponded with an elevation in Cx43 protein expression and phosphorylation. In parallel studies, MAC-T cells were also induced to enter a quiescent state through contact inhibition. Despite a 20-fold reduction in 5-bromo-2'-deoxyuridine and a substantial reduction in intracellular Cx43, contact inhibited MAC-T cells also maintained gap junctions and GJIC. These experiments demonstrate that the maintenance of dye coupling in quiescent mammary cells is correlated with a redistribution of intracellular stores of Cx43.
Animals↗
Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.
To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.
Animals↗
Cellular immunolocalization of occludin during embryonic and postnatal development of the mouse testis and epididymis.
Cellular junctions in the testis and epididymis play crucial roles for the development and maturation of spermatozoa. In the testis, tight junctions between Sertoli cells form a functional blood testis barrier between 10 and 16 days of age, whereas the tight junctional blood epididymal barrier between adjacent epithelial cells is formed between days 18 and 21. In the present study, occludin, a constituent integral membrane protein of tight junctions, was localized by immunofluorescent confocal microscopy in embryonic (days 13.5-18.5), postnatal (days 5-23) and adult (day 70) mouse testes and epididymides to correlate its expression with the onset of tight junctions and eventual formation of these barriers. At embryonic days 13.5 and 16.5, low diffuse cytoplasmic levels of occludin were observed in cells of the testicular cords. By embryonic day 18.5, the level of occludin was still low but appeared as a filiform-like network streaming toward the center of the cord. At postnatal days 5 and 7 immunostaining became more intense and appeared to outline the periphery of Sertoli cells of seminiferous tubules. Postnatal day 14 marked the appearance of an intense, focal band-like localization of occludin at the base of the tubules, correlating with the appearance of a functional blood-testis barrier. By day 23 and in adults, expression of occludin was noted at the base of the tubule appearing as intense, wavy, discontinuous bands similar in appearance irrespective of the stage of the seminiferous epithelium cycle. In the developing epididymis, intense cytoplasmic immunostaining was present in epithelial cells of many epididymal tubules at embryonic day 13.5. By embryonic day 16.5, intense occludin immunostaining appeared along the lateral plasma membranes of epithelial cells, whereas at embryonic day 18.5, immunostaining was punctate and apically located, suggesting the presence of tight junctions by this age; similar immunostaining was noted at postnatal days 5 and 7. In the adult epididymis, distinct punctate apical staining was observed between adjacent principal cells of all epididymal regions except the proximal initial segment, where occludin was found only in association with narrow cells. These results indicate that in the epididymis, the appearance of occludin at apical sites between adjacent epithelial cells occurs during embryonic development suggesting that tight junctions form earlier than in the testis. While occludin was expressed in a similar pattern between Sertoli cells at all stages of the cycle in the adult testis, its expression in the adult epididymis was cell- and region-specific. Taken together these data suggest that different factors regulate occludin expression in the testis and epididymis.
Aging↗
Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy.
The phenomenon of the "bystander effect" (BE) observed in suicide gene therapy studies leads to the intriguing possibility that cytotoxicity can be achieved even in tumor cells that have not themselves been targeted with novel genetic material. There is considerable data suggesting the role of gap junction-mediated intercellular communication (GJIC) in the BE. Transfer of connexin (Cx)-encoding genes, the building blocks of GJIC, has been shown both in vitro and in vivo to increase the BE. Since the loss of GJIC is a common feature of cancer cells, we examined the consequence of GJIC up-regulation on the BE in suicide gene therapy. We used 8-bromo-cyclic-AMP to induce Cx43 and GJIC. In mixing assays, using various proportions of cells containing viral thymidine kinase delivered by an adenoviral delivery system or stably transduced by a retrovirus vector, 8-bromo-cyclic-AMP enhanced the BE of cell killing using ganciclovir. The induction in cell killing was more significant when a low percentage of the cell population was infected, which is the relevant clinical situation. We have demonstrated that this is not due to an effect on infectivity or suicide gene expression. Since decreased GJIC is part of the transformed phenotype, induction of Cxs provides an element of selectivity to suicide gene therapy. Our study adds strength to the rationale to develop clinically tolerable GJ inducers to potentiate the effect of suicide gene therapy via the BE.
8-Bromo Cyclic Adenosine Monophosphate↗
Sex-specific exons control DNA methyltransferase in mammalian germ cells.
The spermatozoon and oocyte genomes bear sex-specific methylation patterns that are established during gametogenesis and are required for the allele-specific expression of imprinted genes in somatic tissues. The mRNA for Dnmt1, the predominant maintenance and de novo DNA (cytosine-5)-methyl transferase in mammals, is present at high levels in postmitotic murine germ cells but undergoes alternative splicing of sex-specific 5' exons, which controls the production and localization of enzyme during specific stages of gametogenesis. An oocyte-specific 5' exon is associated with the production of very large amounts of active Dnmt1 protein, which is truncated at the N terminus and sequestered in the cytoplasm during the later stages of oocyte growth, while a spermatocyte-specific 5' exon interferes with translation and prevents production of Dnmt1 during the prolonged crossing-over stage of male meiosis. During the course of postnatal oogenesis, Dnmt1 is present at high levels in nuclei only in growing dictyate oocytes, a stage during which gynogenetic developmental potential is lost and biparental developmental potential is gained.
Alternative Splicing↗