Gap junction-mediated communication between contiguous cells has been implicated in the regulation of cell proliferation and differentiation. This report describes a new technique to measure cell-cell communication, gap fluorescence redistribution after photobleaching, which is based on the diffusion-dependent return of 6-carboxyfluorescein-mediated fluorescence in a photobleached cell that is in contact with other fluorescently labeled cells. Fluorescence recovery rates are interpreted as dye transport across gap junctions. Results of experiments on normal human fibroblasts and human teratocarcinoma cells show that this technique can measure rapid dye transfer and detect inhibition of communication (between teratocarcinoma cells) by the tumor promoters 12- O -tetradecanoyl-phorbol-13-acetate and the pesticide dieldrin.
Proliferating 3T3 mouse fibroblasts contain higher levels of the lectin carbohydrate-binding protein 35 (CBP35) than do quiescent cultures of the same cells. An immunofluorescence study was carried out with a rabbit antiserum directed against CBP35 to map the cellular fluorescence distribution in a large population of cells under different growth conditions. This cytometric analysis showed that the lectin is predominantly localized in the nucleus of the proliferating cells. In quiescent 3T3 cultures, the majority of the cells lost their nuclear staining and underwent a general decrease in the overall fluorescence intensity. Stimulation ofserum-starved quiescent 3T3 cells by the addition of serum resulted in an increase in the level of CBP35. The percentage of cells showing distinct punctate intranuclear staining reached a maximum at about the same time as the onset of the first S-phase of the cell cycle. All of these results suggest that CBP35 may be a protein whose presence in the nucleus, in discrete punctate distribution, is coordinated with the proliferation state of the cell.In previous studies, we reported the isolation, from 3T3 mouse fibroblasts, of three carbohydrate-binding proteins (CBPs), all of which bind galactose-containing glycoconjugates (1, 2). These were designated CBP35 (Mr 35,000), CBP16 (Mr 16,000), and CBP13.5 (Mr 13,500). A polyclonal rabbit antiserum specific for CBP35 was used to analyze the subcellular localization (3) and tissue distribution (4) of the lectin. These studies revealed that the majority of CBP35 was associated with the cytoplasmic fraction and the nucleus of the 3T3 cell. In addition, CBP35 was identified in adult and embryonic mouse tissues that contained proliferating cell populations (e.g., embryonic liver and skin). In contrast, the lectin was not detected in several adult tissues whose predominant cell population was quiescent (e.g., adult liver and brain).The association of CBP35 with the nucleus and the apparent correlation with proliferating cell populations prompted us to analyze the level of this protein in 3T3 mouse fibroblasts under proliferating and quiescent conditions. In the present communication, we introduce the technique of automated single-cell analysis of fluorescence in anchored cells in tissue culture to document proliferation-dependent expression and nuclear localization of CBP35. This expression of CBP35 and its nuclear translocation are apparently regulated during the G1 phase of the cell cycle. MATERIALS AND METHODSCell Culture and Synchronization. Swiss 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (KC Biological, Lenexa, KS) containing 10% calf serum (Microbiological Associates, Walkersville, MD). Cells cultured at a density less than 5 x 104 cells per cm2 were proliferative and incorporated [3H]thymidine; above this density, the cells remained in a quiescent monolayer state (5). Cells at low density were arrested by removal of serum and maintenance in medium containing 0.2% calf serum for 48 hr. Upon readdition ...
We have previously shown that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress-induced apoptosis, and this involves activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway and expression of the apoptosis repressor with caspase recruitment domain (ARC) protein in the cells. In the present study, we show that up-regulation of the antiapoptotic Bcl-2 family member Mcl-1 is another mechanism critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by small interference RNA (siRNA) rendered melanoma cells sensitive to apoptosis induced by the ER stress inducers thapsigargin and tunicamycin, but this sensitization was partially reversed by siRNA knockdown of PUMA or Noxa, as shown in Mcl-1-deficient melanoma cells. Both PUMA and Noxa were increased by ER stress through transcriptional up-regulation, but only up-regulation of Noxa was dependent on p53, whereas up-regulation of PUMA seemed to be mediated by a p53-independent mechanism(s). Upregulation of Mcl-1 was also due to increased transcription that involved the IRE1A and activating transcription factor 6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway seemed to be necessary for optimal up-regulation of Mcl-1. Taken together, these results reveal the mechanisms of resistance of melanoma cells to apoptosis induction mediated by BH3-only proteins upon ER stress, and identify Mcl-1 as a target for the treatment of melanoma in combination with therapeutics that induce ER stress. [Cancer Res 2008;68(16):6708-17]
Resistance of melanoma cells to chemotherapeutics remains a major obstacle to successful treatment of melanoma once it has spread beyond locoregional sites. We report in this study that activation of the unfolded protein response (UPR) is involved in resistance of melanoma cells to two chemotherapeutic drugs, cisplatin (CDDP) and adriamycin, and this is associated with glucose-regulated protein 78 (GRP78)-mediated inhibition of activation of caspase-4 and -7. The UPR was constitutively activated in cultured melanoma cell lines and fresh melanoma isolates as evidenced by elevated expression levels of the GRP78 protein and the active form of x-box-binding protein 1 messenger RNA. Treatment with CDDP or adriamycin further increased the levels, indicative of induction of endoplasmic reticulum stress and activation of the UPR by the drugs. Inhibition of GRP78 by small-interference RNA (siRNA)-sensitized melanoma cells to CDDP- and adriamycin-induced apoptosis. This was associated with enhanced caspase-4 and -7 activation as siRNA knockdown of the caspases blocked induction of apoptosis. In contrast, overexpression of GRP78 attenuated activation of caspase-4 and -7 and induction of apoptosis by the drugs. CDDP- and adriamycin-induced activation of caspase-4 and -7 appeared to be mediated by calpain activity in that it was blocked by the calpain inhibitors calpeptin and PD150606 even when GRP78 was inhibited by siRNA. These results provide new insights into resistance mechanisms of melanoma cells to CDDP and adriamycin and identify GRP78 as a potential target for enhancing chemosensitivity in melanoma.
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