p53遺伝子欠損によるヒト骨肉腫細胞のシスプラチン耐性獲得

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第1416号，学位授与年月日：平成12年3月31日,学位授与年：200

Reversible differentiation of immortalized human bladder smooth muscle cells accompanied by actin bundle reorganization.

Previous studies have shown that phenotypic modulation of smooth muscle cells (SMCs) plays a pivotal role in human diseases. However, the molecular mechanisms underlying the reversible differentiation of SMCs remain elusive particularly because cultured SMCs that reproducibly exhibit bidirectional phenotypic modulation have not been established. Here we established an immortalized human bladder SMC line designated as hBS11. Under differentiation-inducing conditions, hBS11 cells underwent smooth muscle differentiation accompanied by the robust expression of smooth muscle differentiation markers and isoform-dependent reorganization of actin bundles. The cholinergic receptor agonist carbachol increased intracellular calcium in differentiated hBS11 cells in an acetylcholine muscarinic receptor-dependent manner. Differentiated hBS11 cells displayed contractile properties depending on the elevation in the levels of intracellular calcium. Depolarization of membrane potential triggered inward sodium current in differentiated hBS11 cells. However, differentiated hBS11 cells lost the differentiated phenotype and resumed mitosis when re-fed with growth medium. Our study provides direct evidence pertaining to the human bladder SMCs being able to retain the capacity of reversible differentiation and that the reorganization of actin bundles is involved in the reinstatement of contractility. Moreover, we have established a human SMC line retaining high proliferating potential without compromising differentiation potential

A23187-induced contraction of immortalized human bladder smooth muscle cells.

<p>(A) hBS11 cells were preloaded with SiR-actin (100 nM) for 2 h on day 3 or 9 of differentiation culture and then cultured for another 3 days in pmDM. Next, the cells were treated with a calcium ionophore A23187 (5 μM) on day 6 (upper row) or 12 (lower row) of differentiated culture. The cells were sequentially observed using epifluorescence microscopy and time-lapse recordings at 5 s intervals. Small arrows represent contracted actin bundles. Incubation time before and after stimulation with A23187 is shown at the upper panel corners. Scale bar, 10 μm. (B) Cells were cultured in pmDM for 12 days and stimulated with (f-j) or without (a-e) A23187 (5 μM) for 10 min. Next, the cells were subjected to filamentous actin staining with Alexa 546-conjugated phalloidin (red in b, d, g and i) and immunostaining analysis with antibodies for α-SMA (green). Images of the same fields are shown in a-c, d-e, f-h and i-j, respectively. Arrow heads represent thickened knob-like actin bundles. Nuclei were stained with DAPI (blue). Scale bars, 100 μm for (a-c and f-h) and 20 μm for (d,e,i and j). (C) hBS11 cells were cultured in pmDM for 7 days and stimulated with A23187 (5 μM) for 45 min. Next, the cells were subjected to immunostaining analysis with antibodies for α-SMA (green) and β-CYA (red). The phase contrast image (a) and merged image (b) of c and d are shown. The cells that were intensely stained with α-SMA antibody (arrow heads) exclusively shrank. In contrast, the intact spreading cells (arrows) contained β-CYA-positive and α-SMA-negative bundles. Scale bar, 100 μm.</p

Phenotypic modulation of human bladder smooth muscle cells.

<p>(A) Structural changes of a bladder between contraction for expelling and expansion for storage of urine. Arrows represent the direction of intraluminal pressure. (B) A schematic figure for bidirectional phenotypic modulation of human bladder smooth muscle cells. Detailed explanations and discussion for reversible differentiation and isoform-dependent reorganization of actin bundles are outlined in the main text. (C) A schematic figure for the phenotypic modulation of vascular smooth muscle cells. Dedifferentiated SMC is a collective term of a variety of SMC subtypes. Dedifferentiation from contractile phenotype to synthetic phenotype is often irreversible or partially reversible.</p

Gene ontology analysis of upregulated genes in differentiated hBS11 cells.

<p>Gene ontology analysis of upregulated genes in differentiated hBS11 cells.</p

Calcium increase in immortalized human bladder smooth muscle cells.

<p>(A) Differentiated hBS11 cells were preloaded with a calcium sensitive dye Fluo-4 AM, and then stimulated with a cholinergic agonist carbachol (1 mM). The cells were observed under epifluorescence microscopy. Fluorescent images correspond to the frames 2, 5, 10, 20, 31, and 37 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186584#pone.0186584.s012" target="_blank">S3 Video</a>. Scale bar, 10 μm. (B) Differentiated hBS11 cells were treated as described in (A). The cells were observed under phase contrast and epifluorescence microscopy. The phase contrast image was taken before stimulation with carbachol. Fluorescent images correspond to the frames 28 and 34 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186584#pone.0186584.s013" target="_blank">S4 Video</a>. A circle represents a cell-to-cell contact region between neighboring cells named #1 and #2 in a phase contrast image before carbachol stimulation (left panel). Calcium signaling was conducted between neighboring cells (middle and right panels). (C) hBS11 cells were preloaded with Fluo-4 AM for 1 h on day 14 or 19 of differentiation culture and then stimulated with a cholinergic agonist carbachol (50 μM) for 30 s. Digital fluorescent imaging was obtained using a two-photon confocal microscope. ΔF/F₀ represents percent changes in the fluorescence intensity over resting levels. (a) Effect of the muscarinic receptor antagonist atropine (5 μM) on carbachol-induced intracellular Ca²⁺ elevation. Horizontal bar represents the period of exposure to carbachol. F/F₀ of hBS11 cells before (open circles), during the treatment of atropine (red circles), and after washing off atropine (blue circles) are shown. (b) Pooled data regarding the effect of atropine on carbachol-induced intracellular Ca²⁺ elevation. Atropine treatment significantly blocked the Ca²⁺ elevation. Statistical significance (p-value) was estimated using the multi-comparison Dunnett’s test (n = 8). Each dashed line connecting open circles represents data obtained from the same cells. Each bar represents average and standard error of mean. (D) hBS11 cells were cultured for 14 days in pmDM and preloaded with Fluo-4 AM. Next, the medium was switched to a calcium-deleted Krebs-Ringer solution supplemented with 90 mM KCl. The cells were sequentially observed using epifluorescence microscopy and time-lapse recordings with a 30-second interval. Incubation time before and after stimulation with calcium (2.8 mM) is shown at the upper panel corners. Scale bar, 10 μm.</p