Park Y M, Mivechi N F, Auger E A, Hahn G M
Stanford University School of Medicine, Department of Radiation Oncology, CA 94305-5468.
Int J Radiat Oncol Biol Phys. 1994 Jan 1;28(1):179-87. doi: 10.1016/0360-3016(94)90156-2.
The differences in the heat shock gene regulation between the RIF-1 cell line and its heat resistant derivative TR4 are further characterized.
In vitro gel retardation assays were used to assess the presence of activated heat shock transcription factor in the two cell lines. The levels of the heat-inducible HSP 70.1, the constitutive HSC 70, the germ line-specific HSP 70.2, and the HSP 28 mRNAs in both untreated and iso-heated RIF-1 and TR4 cells were determined using the polymerase chain reaction coupled with the reverse transcriptase reaction. Induction and decay of induced heat shock protein synthesis was measured by 35S-methionine labeling of proteins.
Unheated TR4 cells display characteristics of heat shocked RIF-1 cells. TR4 cells have a constitutively activated heat shock transcription factor and elevated levels of the HSP 70.1, HSC 70, and the HSP 28 mRNAs. Upon an equal heat dose of 45 degrees C, 15 min, the TR4 cells exhibited a more rapid onset in heat shock mRNA and protein induction than did the RIF-1 cells. During the recovery from heat shock, the activated heat shock transcription factor and the induced HSP70 mRNAs decayed more slowly in the TR4 cells, although the protein synthesis pattern of the TR4 cells returned to control levels more rapidly following heat shock than did protein synthesis of the RIF-1 cells.
Unheated TR4 cells are similar to heat shocked RIF-1 cells at the transcriptional level. Induced HSP70 expression is modulated by the severity of the heat treatment (or the degree of heat damage) perceived by the cells rather than by the absolute heat dose given. We propose that the unheated TR4 cells are locked into the "ON" state of the heat shock response.
进一步表征RIF-1细胞系与其耐热衍生物TR4之间热休克基因调控的差异。
体外凝胶阻滞试验用于评估两种细胞系中活化的热休克转录因子的存在情况。使用聚合酶链反应结合逆转录反应，测定未处理和等热处理的RIF-1和TR4细胞中热诱导型HSP 70.1、组成型HSC 70、种系特异性HSP 70.2和HSP 28 mRNA的水平。通过蛋白质的35S-甲硫氨酸标记来测量诱导的热休克蛋白合成的诱导和衰减。
未加热的TR4细胞表现出热休克RIF-1细胞的特征。TR4细胞具有组成性活化的热休克转录因子，且HSP 70.1、HSC 70和HSP 28 mRNA水平升高。在45℃、15分钟的同等热剂量下，TR4细胞比RIF-1细胞在热休克mRNA和蛋白诱导方面表现出更快的起始速度。在从热休克中恢复的过程中，活化的热休克转录因子和诱导的HSP70 mRNA在TR4细胞中的衰减更慢，尽管TR4细胞的蛋白质合成模式在热休克后比RIF-1细胞的蛋白质合成更快地恢复到对照水平。
未加热的TR4细胞在转录水平上与热休克的RIF-1细胞相似。诱导的HSP70表达受细胞感知的热处理严重程度（或热损伤程度）调节，而非所给予的绝对热剂量。我们提出未加热的TR4细胞被锁定在热休克反应的“开启”状态。
