## 3 The Effect of RF Exposure on _in Vitro_ Models: Finding the Pathways
### Intracellular Pathways: The Usual Suspects  
Early _in vitro_ studies of RF-EMF were mostly designed to study the toxic effect of RF on cell. Some of them aimed at clarifying the intracellular pathways that would sustain induced DNA damage and cellular fate. For example, [PERSON] [28,29] demonstrated a genotoxic effect on amniotic cells exposed for 24 h (SAR 0.25 W/kg) to GSM-900 MHz. However, no direct cytogenetic effect was detected after exposure. Recently, replicated dosing of a wider range of SAR (up to 4 W/kg) and similar experiments indicated that exposure did not affect the intracellular stress-related p53 pathway. p53 is an important checkpoint system which guards against genomic instability by inducing both arrest of the cell cycle and apoptosis. Previous data on short exposure of 900 MHz in lymphoblastic leukemia indicated activation of p53-related pathways. However, longer continuous exposure determined silencing of pro-apoptotic signals and activation of pro-surviving genes such as _Bcl-2_ and _Ras_ and _Ak1_. This dual effect can be reconciled as an early self-defense response triggered by DNA damage that confers to the survivor cells a further advantage to survive and proliferate [30]. In this case, the authors reported both genotoxic damage and changes in gene expression levels. To draw a complete picture of lymphoblastic leukemia response to RF exposure, further investigation was recently carried out by microarray analysis on cancer cells. [PERSON] and coworkers [31] confirmed that high frequency EMFs affect cellular systems by acting as genotoxic agents. Significant changes in expression levels of genes involved in DNA repair, cell cycle arrest, apoptosis, chromosomal organization, and angiogenesis were observed after both short- and long-term 900 MHz EMF exposure. DNA repairing machinery was found to be dually affected with activation of p53-related genes and down-regulation of down-strain effectors like BRCA2, XRCC3, and RFC1. Apoptotic signaling was affected by RF exposure, as APAF1 was found to be strongly over-expressed, while FASL and CASP8 were found to be down-regulated. Another component of \"caspase\" cascade, CASP10, was found to be up-regulated only at an early time. It appears that RF, acting as a genotoxic agent, quickly induces DNA damage by activating early on cell death effectors of apoptotic response. With respect to aggressiveness of the tumor cells, altered gene expression was detected for angiogenesis, differentiation and regulation of the cytoskeleton. In relation to genes involved in angiogenesis signaling, down-regulation of BAI1 and TNFSF15 was observed. Similarly, VEGFA, which acts in the permeability of endothelial cells, and FLT4 were inhibited at both MW-EMF exposure time, while EPO, IL8, STAT5B and VAV2, PGF, HPSE appear to be strongly activated. It appears therefore that in lymphoblastic leukemia cells, the 900 MHz MW-EMF may act as a double target negative regulator of genes: it affects the control of chromosomal organization and induces inhibition of angiogenesis that leads to tumor progression and metastatic transformation. It is worth mentioning that several previous microarray analyses carried out after RF exposure failed to detect any significant changes in the gene expression in normal peripheral [32] or CNS glial cells [33] or cancer cell models. To add complexity to the picture, [PERSON] and co-workers [22], comparing the proteomic in Jurkat T-cells and human fibroblast, indicated that a strict relation between metabolic state and responsiveness to RF-EMF exists that implies a higher sensitivity of growing organisms. Moreover, these authors hypothesize that disturbances of hydrogen bonds might play a crucial role to sustain alteration in protein profile after RF active cells to determine its bioeffects. Cell architectural elements, such as a membrane or a cytoskeleton, can be considered a possible target for EMF radiation damage.