Our results show that THz irradiation of mMSCs can cause specific catalytic changes in cellular function that are closely related to the gene expression and differentiation state. The strictly controlled experimental environment indicates minimal temperature changes and the absence of any discernable response to heat shock and cellular stress, thus implying a non-thermal cellular response to the applied low power THz stimulus. Our RT-PCR experiments and gene expression survey suggest that some genes in the irradiated mMSCs cultures are activated, while others are suppressed. Importantly, the different irradiation conditions result in dissimilar regulation of Car3, Ctfg, Gem, Nfe2l2, and Slco4a, indicating that gene expression is dependent on the parameters and duration of THz irradiation. Interestingly, even 2 hours of irradiation are sufficient to cause differential changes in gene expression for certain active genes (e.g., Slco4a1).
In our previous work12,13
, we irradiated mMSC cultures 120 hours after cell treatment with medium that induces differentiation toward adipose phenotype, and observed activated expression of marker genes for adipocytes as well as lipid droplet-like inclusions in the cellular cytoplasm. Here, we report changes in gene expression of the adipocyte markers, but lack of morphological changes in mMSCs that were irradiated (at the same THz conditions) only 48 hours after the treatment with the same differentiation medium (i.e.
, presumably at an earlier differentiation state). Hence, irradiating stem cells further in their differentiation program (viz.
, ones irradiated 120 hours after treatment) results in noticeable morphological changes that are not displayed in cells irradiated at an earlier differentiation stage. This observation suggests that the effect of THz irradiation depends on the differentiation state of the stem cells.
One significant concern surrounding these results is that no comprehensive model can explain the effect of THz radiation. Our suggestion22,23
is that THz radiation may affect gene expression by interacting (in a nonlinear resonant way) with the hydrogen bonds of biomacromolecules, whose vibrational frequencies are in the THz range41,42
, by perturbing the conformational dynamics of double-stranded DNA or by affecting protein-DNA binding. This suggestion is based on prior work37,43,44
that establishes a strong relationship between breathing dynamics of double-stranded DNA and cellular function36,38
, as well as between DNA breathing and protein-DNA binding in some cases39,40,45
. Our suggestion is also supported by the fact that millimeter wave radiation are capable of melting DNA oligomers by disrupting only intramolecular hydrogen bonds46
. Furthermore, our experimental results are consistent with this proposed explanation, since some overexpressed genes have high propensity for DNA breathing. Nevertheless, the observed results may be due to other effects such as changes in the cell's membrane potential47
, or protein-protein interactions48
, or in the protein conformation itself49
. Further molecular level experimental investigations of THz radiation's ability to induce specific openings of double-stranded DNA and changes in protein-DNA binding are needed to fully determine the validity of our hypothesis.
Finally, the performed microarray survey and RT-PCR experiments reveal a small group of differentially expressed genes in all three experimental irradiation scenarios. Even this small group of affected genes shows opposing effects of THz radiation on gene expression, and points toward the need for comprehensively cataloging genes that can be affected under specific THz irradiation parameters. The generation of such a catalogue is not a simple task, and it will involve a large-scale experimental effort with potentially tens of replicates irradiated under a large variety of conditions, taking into account (at least) parameters such as: amplitude of the THz field, narrow frequency intervals, and exposure duration. Nevertheless, the generation of such a catalogue will be instrumental in opening the door to using THz radiation for non-contact control of cellular gene expression. We are positive that additional efforts, such as including protein and functional readouts as well as in vivo and ex vivo investigations of the effect of THz radiation are needed to validate, generalize, and obtain stronger insight into the effect of the millimeter wave radiation on gene expression. Further insight into how the THz radiation can alter: the DNA breathing; or cell membrane potential; protein-DNA or protein-protein binding; or in general conformations of biomacromolecules (e.g., the local bending), is needed to shed light on the mechanisms of the THz effect on biological matter.