Zölzer Friedo, Streffer Christian
Institut für Medizinische Strahlenbiologie, Universitätsklinikum Essen, Essen, Germany.
Int J Radiat Oncol Biol Phys. 2002 Nov 1;54(3):910-20. doi: 10.1016/s0360-3016(02)02963-2.
It is well known that the radiosensitivity of tumor cells can be significantly reduced under hypoxic conditions. However, most of the reports in the literature refer to an experimental setup in which the supply of oxygen is kept low for a short period of time only. In tumors, chronic hypoxia would seem to be the more typical situation, because of an insufficient vascularization and the limited diffusion of oxygen into the tissue. Under such conditions, certain changes in the proliferation patterns of tumor cells, in which the cell cycle checkpoint protein p53 seems to play a role, have been shown to occur. We therefore decided to study radiosensitivity and cell cycle progression under conditions of chronic hypoxia in several human tumor cell lines differing in their p53 status.
Four human tumor cell lines (melanomas Be11 and MeWo and squamous carcinomas 4197 and 4451) were incubated for 3 h, 24 h, and 72 h under either oxic or hypoxic conditions and subsequently exposed to graded doses of X-rays. In some cases, cells were kept under hypoxia for the same periods of time, but then reoxygenated immediately before irradiation. Cell survival was assessed with the usual colony formation assay, and cell cycle distributions were determined by two-parameter flow cytometry after labeling with bromodeoxyuridine (BrdU).
As expected, the oxygen enhancement ratio at 3 h was 2.0 or more in all cases. Differences, however, became evident with longer incubation times. At 24 h, the sensitivity of cells kept under hypoxic conditions both before and during irradiation was practically unchanged with cell lines Be11, 4197, and 4451, but clearly increased with MeWo. This resulted in an oxygen enhancement ratio of only 1.1 for the latter cell line when the sensitivity of aerated cells was used as reference. Cells kept under hypoxia for 24 h and reoxygenated shortly before irradiation, however, also showed an increase in sensitivity, so that the oxygen enhancement ratio based on differences in irradiation atmosphere alone was still around 2.0. At 72 h, the two p53 wild-type cell lines were not available for experiments, because they quickly degenerated under hypoxic conditions. Both mutant cell lines now showed similar results, the sensitivity being increased with irradiation under continued hypoxia as well as after reoxygenation. The oxygen enhancement ratios with reference to aerated cells were 1.3 and 1.5 for MeWo and 4451, respectively. Flow cytometric measurements after labeling with BrdU revealed that in all cell lines, the fraction of active S-phase cells during incubation tended to decrease under hypoxic conditions. Only in the p53 mutant cell lines, however, was this accompanied by an increase of the percentage of S-phase cells that were not actively incorporating BrdU.
It is suggested that these quiescent cells in the S-phase compartment develop because of a general breakdown of cellular energy metabolism. In the p53 mutant cells, this may lead to a cessation of cell cycle progression in all phases alike, because checkpoint control has been lost; p53 wild-type cells, on the other hand, settle down preferentially in G(1) under the same conditions. Independently of the p53 status, however, energy depletion may be the cause of a decreased ability to cope with radiation damage and thus the cause of the observed increase in radiosensitivity. This would become more easily apparent in the p53 mutant cell lines, because they are less sensitive than the p53 wild types to hypoxia as such.
众所周知，在缺氧条件下肿瘤细胞的放射敏感性会显著降低。然而，文献中的大多数报道涉及的实验设置是仅在短时间内保持低氧供应。在肿瘤中，由于血管生成不足以及氧气向组织内的扩散受限，慢性缺氧似乎是更典型的情况。在这种条件下，已显示肿瘤细胞的增殖模式会发生某些变化，其中细胞周期检查点蛋白p53似乎发挥了作用。因此，我们决定研究在慢性缺氧条件下，几种p53状态不同的人肿瘤细胞系的放射敏感性和细胞周期进程。
四种人肿瘤细胞系（黑色素瘤Be11和MeWo以及鳞状细胞癌4197和4451）在有氧或缺氧条件下分别孵育3小时、24小时和72小时，随后接受分级剂量的X射线照射。在某些情况下，细胞在缺氧条件下保持相同的时间，但在照射前立即复氧。用常规的集落形成试验评估细胞存活率，并用溴脱氧尿苷（BrdU）标记后通过双参数流式细胞术测定细胞周期分布。
正如预期的那样，在所有情况下3小时时的氧增强比为2.0或更高。然而，随着孵育时间延长差异变得明显。在24小时时，对于细胞系Be11、4197和4451，在照射前和照射期间都保持在缺氧条件下的细胞的敏感性实际上没有变化，但对于MeWo明显增加。当以通气细胞的敏感性作为参考时，后一种细胞系的氧增强比仅为1.1。然而，在缺氧条件下保持24小时并在照射前不久复氧的细胞也显示出敏感性增加，因此仅基于照射环境差异的氧增强比仍约为2.0。在72小时时，两种p53野生型细胞系无法用于实验，因为它们在缺氧条件下迅速退化。现在两种突变细胞系显示出相似的结果，在持续缺氧照射以及复氧后敏感性均增加。相对于通气细胞，MeWo和4451的氧增强比分别为1.3和1.5。用BrdU标记后的流式细胞术测量显示，在所有细胞系中，孵育期间活跃的S期细胞比例在缺氧条件下趋于下降。然而，仅在p53突变细胞系中，这伴随着未积极掺入BrdU的S期细胞百分比的增加。
提示S期隔室中的这些静止细胞是由于细胞能量代谢的普遍崩溃而产生的。在p53突变细胞中，这可能导致细胞周期在所有阶段都停止进展，因为检查点控制已经丧失；另一方面，p53野生型细胞在相同条件下优先停滞在G(1)期。然而，无论p53状态如何，能量耗竭可能是应对辐射损伤能力下降的原因，因此也是观察到的放射敏感性增加的原因。这在p53突变细胞系中更容易显现，因为它们本身比p53野生型对缺氧更不敏感。
