Abstract
Cell shape and architecture are determined by cell-extracellular matrix interactions and have profound effects on cellular behavior, chromatin condensation, and tumor cell resistance to radiotherapy and chemotherapy. To evaluate the role of chromatin condensation for radiation cell survival, tumor cells grown in three-dimensional (3D) cell cultures as xenografts and monolayer cell cultures were compared. Here, we show that increased levels of heterochromatin in 3D cell cultures characterized by histone H3 deacetylation and induced heterochromatin protein 1alpha expression result in increased radiation survival and reduced numbers of DNA double strand breaks (DSB) and lethal chromosome aberrations. Intriguingly, euchromatin to heterochromatin-associated DSBs were equally distributed in irradiated 3D cell cultures and xenograft tumors, whereas irradiated monolayer cultures showed a 2:1 euchromatin to heterochromatin DSB distribution. Depletion of histone deacetylase (HDAC) 1/2/4 or application of the class I/II pharmacologic HDAC inhibitor LBH589 induced moderate or strong chromatin decondensation, respectively, which was translated into cell line-dependent radiosensitization and, in case of LBH589, into an increased number of DSBs. Neither growth conditions nor HDAC modifications significantly affected the radiation-induced phosphorylation of the important DNA repair protein ataxia telangiectasia mutated. Our data show an interrelation between cell morphology and cellular radiosensitivity essentially based on chromatin organization. Understanding the molecular mechanisms by which chromatin structure influences the processing of radiation-induced DNA lesions is of high relevance for normal tissue protection and optimization of cancer therapy.
Highlights
Cell shape and architecture have profound impact on cellular behavior [1]
To gain first insight into the consequences of 3D-induced heterochromatin formation for residual DSBs (rDSB) after ionizing radiation, we examined γH2AX colocalization with HP1α in HP1αEGFP transfectants and in A549 xenograft tumors. γH2AX foci were defined as euchromatic foci (EC), and γH2AX foci overlapping with or in vicinity to HP1α were defined as heterochromatic foci (HC; Fig. 3A; Supplementary Fig. S11)
We show that increased radiation survival in a 3D microenvironment results from an elevated amount of heterochromatin, a differential dissemination of euchromatin to heterochromatin–associated double strand breaks (DSB) in 3D and in vivo versus 2D and a lower number of lethal chromosomal aberrations
Summary
Models of normal and transformed cells impressively showed that three-dimensional (3D) growth in an extracellular matrix (ECM) substantially modifies gene and protein expression, survival, proliferation, differentiation, and metabolism compared with conventional monolayer cell cultures [2,3,4]. These findings suggest that cell morphology fundamentally determines tissue homeostasis and cellular responsiveness to external stress signals in a microenvironment-specific context. DSBs induced by ionizing radiation are life-threatening DNA lesions Their inaccurate and inefficient repair results in chromosomal aberrations, loss of genome integrity, and cell death [26, 27]. Depletion of HDAC1/2/ 4 or application of the class I/II pharmacologic HDAC inhibitor LBH589 induced moderate or strong chromatin decondensation, respectively, which translated into enhanced cell line–dependent radiation sensitivity and, in the case of LHB589, into an increased number of DSBs
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