Sit K H, Paramanantham R, Bay B H, Wong K P
Department of Anatomy, Faculty of Medicine, National University of Singapore, Kent Ridge.
Anat Rec. 1994 Dec;240(4):456-68. doi: 10.1002/ar.1092400404.
The early stages of apoptosis (programmed cell death) are said to be characterized by internucleosomal DNA fragmentation and "condensation of the cytoplasm" in which cells round up, detach, and increase in density. We studied the causation of apoptotic rounding.
Human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts derived from serum growth factor deprivation. Cell-by-cell analysis using the Coulter EPICS PROFILE II flow cytometer studied 1) the cell cycle from propidium iodide-DNA bindings, 2) uptake of neutral red (NR) dye, a viable cell marker, and 3) cytosolic pH (pHi) modulations from 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) fluorescence ratios with NH4Cl prepulsing and forward scatter bitmapping of cell surface area. Morphometric studies were done in the Quantimet 570 image analyser. Uptake of trypan blue, neutral red, and 2 million mol.wt fluoresceinated dextrans was studied by light microscopy. Cytological profiles were examined in light microscopy and transmission and scanning electron microscopy.
Three days of serum growth factor deprivation caused confluent flat substrate-attached cells to retract and round up, tethering tenuously to the substrate via thin microvillus attachments only. Ninety percent of cell surface area was lost with this flat-to-round change. There was high trypan blue staining with total loss of proliferative potential, and the entire genome was just fragmented DNA making up the solitary Ao (apoptotic) peak in cell cycle profiles. However, these rounded apoptotic cells also internalized huge 2 million mol.wt dextran particles and impermeant neutral red which is an established viable cell marker. The rounded apoptotic cells had an intensely acidic (pH 5.6) cytosol and therefore a steep [H+]i/[H+]o gradient promoting proton extrusion. The pHi upshifted dynamically upon acidification, recovering and even exceeding resting level by a whole pH unit. Surface area reduction occurred concomitantly in real time with pHi upshifts in these apoptotic cells. Acidification and recovery in apoptotic cells also produced enhanced uptake of neutral red. Cytological profiles showed abundant large endocytic channels and endosomes in the rounded apoptotic cells.
Gross surface area reduction with evidence of distinctive endocytic activity including uptake of huge 2 million mol.wt dextran particles suggested large channel endocytic internalization as a causal factor in apoptotic rounding, in common with rounding in M-phase and interphase cells with pHi upshifting where concomitant surface area reduction and uptake of impermeant particles were similarly demonstrable. The reduction in size of the cell envelope, together with consequential concentration pressures, could account for the observed rise in cell density and shrinkage in cell size. As a symptom of continual pHi upshifting, apoptotic rounding appears to be a recovery-associated response rather than a direct consequence of the disruptive forces causing its death.
细胞凋亡（程序性细胞死亡）的早期阶段据说具有核小体间DNA片段化和“细胞质浓缩”的特征，即细胞变圆、脱离并密度增加。我们研究了凋亡变圆的原因。
将正常单层培养的人Chang肝细胞与因血清生长因子剥夺而凋亡的细胞进行比较。使用库尔特EPICS PROFILE II流式细胞仪进行逐个细胞分析，研究1）碘化丙啶 - DNA结合的细胞周期，2）中性红（NR）染料的摄取，一种活细胞标记物，以及3）通过2',7'-双（2 - 羧乙基）-5（和 - 6）-羧基荧光素（BCECF）荧光比率与氯化铵预脉冲以及细胞表面积的前向散射位图来调节细胞溶质pH（pHi）。在Quantimet 570图像分析仪中进行形态计量学研究。通过光学显微镜研究台盼蓝、中性红和200万分子量荧光素化葡聚糖的摄取。在光学显微镜、透射电子显微镜和扫描电子显微镜下检查细胞学特征。
血清生长因子剥夺三天导致汇合的扁平贴壁细胞回缩并变圆，仅通过细小微绒毛附着微弱地附着于基质。这种从扁平到圆形的变化使细胞表面积损失了90%。台盼蓝染色阳性，增殖潜能完全丧失，整个基因组只是碎片化的DNA，在细胞周期图谱中形成单独的凋亡（Ao）峰。然而，这些圆形凋亡细胞也内化了巨大的200万分子量葡聚糖颗粒和不透性的中性红，而中性红是一种公认的活细胞标记物。圆形凋亡细胞具有强烈酸性的细胞溶质（pH 5.6），因此具有陡峭的[H⁺]i/[H⁺]o梯度促进质子外排。酸化时pHi动态上移，恢复甚至超过静止水平达整个pH单位。这些凋亡细胞表面积减少与pHi上移同时实时发生。凋亡细胞的酸化和恢复也导致中性红摄取增加。细胞学特征显示圆形凋亡细胞中有丰富的大型内吞通道和内体。
明显的表面积减少以及独特的内吞活性证据，包括摄取巨大的200万分子量葡聚糖颗粒，表明大通道内吞内化是凋亡变圆的一个因果因素，这与M期和间期细胞变圆时类似，此时pHi上移，同时表面积减少和不透性颗粒摄取也同样明显。细胞膜大小的减小以及随之而来的浓缩压力，可以解释观察到的细胞密度增加和细胞大小缩小。作为持续pHi上移的一种表现，凋亡变圆似乎是一种与恢复相关的反应，而不是导致其死亡的破坏力量的直接后果。
