Energy Deposition leads to Increase, Cell Proliferation

The biological plausibility of the relationship between deposited energy leading to increased proliferation of cells is moderately supported by the literature. The deposition of energy, such as that from ionizing radiation, starting at doses of <0.5 Gy for in vivo and >2 Gy for in vitro has been shown to induce changes in cell proliferation (Uwineza et al., 2019; Hamada & Sato, 2016; Hamada, 2017a; Ainsbury et al., 2016). Rabbit, human, and rat models and many cell types have been used to support this connection. Increased cell proliferation is induced via energy deposition through a poorly understood mechanism of signalling changes and gene expression alterations. The evidence supporting the connection spans multiple life stages and sexes, though no observable differences can be delineated between the groups (Markiewicz et al., 2015; Fujimichi & Hamada, 2014; Worgul et al., 1986; Richards, 1966; Barnard et al., 2022; Riley et al., 1989; von Sallmann, 1952; Soderberg et al., 1986; Ramsell & Berry, 1966; Treton & Courtois, 1981). 

One of the main cell-related changes that can occur as a result of energy deposition is changes in gene expression. This is an event that then causes cell proliferation to increase. Energy deposition via stressors, such as ionizing radiation, increase cell proliferation through the inactivation of tumor suppressor genes and oncogene activation. Activated oncogenes can increase cell proliferation while the inactive suppressor genes are unable to regulate this change. Cyclin-D1, an oncogene protein, has been linked to shortened times between G1 and S stages of the cell cycle when found in excess in the cell (Reynolds & Schecker, 1995).  

Other events also occur within the organism that contribute to increased proliferation following energy deposition. Alterations in cell growth factor genes such as MAPK1, as a result of deposited energy, can also cause increased proliferation rates in LECs (Vigneux et al., 2022). The mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 30-kinase (PI3K/AKT) signalling pathways are anti-apoptotic, with the former being essential for G2 checkpoint arrest (Hein et al., 2014). PI3K/AKT signalling is altered by growth factors and hindered by tumor suppressors (Dillon et al., 2007). MAPK/ERK and PI3K/AKT signalling pathways are both activated by energy deposition, such as ionizing radiation, and this has been shown to increase cell proliferation in human lung fibroblast cells. (Liang et al., 2016). PI3K is turned on by the increased growth factor levels and suppressor gene downregulation (Lee & Muller, 2010). This then signals down the pathway until it reaches AKT. AKT turns on the MAPK/ERK pathway, which induces increased cell proliferation, though the exact mechanism by which this happens is still uncertain (Liang et al., 2016). 

The empirical evidence relating to this KER strongly supports the relationship between the deposition of energy and increased cell proliferation. A variety of cell growth changes have been used to measure this relationship, including mitotic activity and the presence of proliferative markers. The data comes from a mix of in vivo and in vitro studies on mice, rats, rabbits, and human, with space-related radiation types such as γ-rays, UV, or X-rays (Markiewicz et al., 2015; Bahia et al., 2018; Fujimichi & Hamada, 2014; Worgul et al., 1986; Richards, 1966; Barnard et al., 2022; Riley et al., 1989; von Sallmann, 1952; Soderberg et al., 1986; Andley et al., 1994; Ramsell & Berry, 1966; Treton & Courtois, 1981).  

Dose Concordance 

High evidence exists to support dose response between energy deposition and increased cell proliferation. There is much available evidence to support this relationship using the lens of the eye. 

Mitotic activity changes can be observed in vivo starting at 2 Gy neutron and 3 Gy X-ray exposures. With X-ray doses up to 1 Gy, no mitotic change in lens epithelial cells is observed. At 3 Gy, there is a ~0.5%/day change in mitosis, and this increases linearly to ~2.25% at 10 Gy (Riley et al., 1989). With a 3.5 Gy and 10 Gy dose, mitotic lens cells increased to about ~35% and ~45% relative to control treatment groups, respectively. Similarly, lens cells irradiated with 1 Gy argon ions had ~30% of cells undergoing mitosis (Worgul et al., 1986). In this study, mitotic activity in lens epithelial cells reached a peak at ~3 fold of the normal range after single 2 Gy neutron irradiation, in contrast to ~2-fold after single 4 Gy X-ray irradiation. When doses are administered in fractions, mitotic activity reached its peak at 2x normal range after neutron exposure and 3x after X-ray exposure. Whole lenses irradiated with neutrons showed a 6-fold increase in proliferating cells compared to X-irradiated cells. Irradiation of smaller areas wthin lens cells resulted in a smaller increase in mitotic activities (Richards, 1966). In another study, it was shown that lens epithelium were displaying 69% higher levels of proliferating cells than the control following an in vivo 10 Gy X-ray exposure (Ramsell & Berry, 1966). Unscheduled synthesis, as measured by the number of grains in non-S phase cells and defined as an excess in the normal duplication of the genome prior to mitosis, increased in the lens epithelium to 8.3x control following in vivo exposure to 6 kJ/m2 of UV (Soderberg et al., 1986). In vitro [3H]-thymidine incorporation in lens epithelial cells following 250 J/m2 UV was 6.46x control levels (Andley et al., 1994). 72 weeks post 50 J/m2 UV exposure, the epithelio-distal region lens cells reached 5.3x control’s grains/nuclei and the epithelio-central region cells reached 11x control using an in vitro model. The treatment group for the mitotic zone reached 2.4x control (Treton & Courtois, 1981). 

An increase in the in vivo incorporation of the cell proliferation marker EdU was observed in lens cells following X-ray exposure of 0.05 Gy or higher. This then decreased following exposure to 1 Gy. Cells in the lens transition zone had 2x more EdU+ cells following 0.05 Gy, compared to the control. This increased linearly to 13x control levels following 0.25 Gy. Lens cells in the germination zone increased 5x when exposed to 0.25 Gy. Cells in both zones returned to control levels at doses of 1 Gy and above (Markiewicz et al., 2015).  

Indirect assessment of cell proliferation through measurement of cell number, following exposure of immortalized human lens epithelial cells to X-rays from 0.01- 5 Gy showed the number of cells increased at 0.01 Gy with levels 1.5x above the control. At 0.5 Gy and above, the in vitro lens epithelial cell number decreased towards control levels (Bahia et al., 2018). Another in vitro study examined the amount of the epithelium area covered in the lens, finding that it remained consistent with control groups up to 2 Gy, and increased significantly at doses ≥2 Gy, remaining above 1.5x control. The number of colonies considered large (mean control area + 2 standard deviations) also increased at doses above 2 Gy after remaining near control levels at lower doses (Fujimichi & Hamada, 2014). 

Time Concordance 

High evidence exists to support time response between energy deposition and increased cell proliferation. Most studies show increased proliferation occurring at varied time points following the initial incidence of energy deposition, with most evidence being from high (≥2 Gy) doses of ionizing radiation exposure. 

 In examining, mitotic activities within in vivo lens cells irradiated with 1 Gy 40-Ar and 3.5 Gy X-ray, a suppression of proliferating cells was observed at 1-day post-irradiation, followed by an overshoot from control levels as early as day 3 and then a decline ~3 weeks post-irradiation (Worgul et al., 1986). Mitotic activity in another in vivo study reached peak at ~ 13 days post following irradiation from a single 2 Gy neutron source and 7 days after a single 4 Gy X-irradiation for lens epithelial cells. Activity reached normal or below normal range 14 days to ~1-month post-exposure. When administered in fractioned doses, activity peaked at 5-7 days post neutron exposure and 3-5 days post X-irradiation (Richards, 1966). Lens cell mitotic levels at 29 days post in vivo irradiation were ~15% higher than seen at time zero, following 1.25 Gy neutron exposure (Riley et al., 1989). After in vivo exposure to 2 Gy neutron radiation, there was an increase in mitotic activity starting within 4 hrs post exposure and occurring in up to ~45% of the lens cell population. Cells exposed to 10 Gy X-rays showed a similar trend, but only in up to ~27.5% of cell population (Riley et al., 1988). There was no response to the in vitro 15 Gy X-ray exposure up until 6 days post-irradiation, when the mitotic response reached 150 mitoses/lens epithelial cell. 12 days post-irradiation, the mean score was about 345 mitoses/cell while control remained at 0 mitoses/cell for the whole testing period (von Sallmann, 1952).  

Essentiality 

Moderate evidence exists to support the essentiality of energy deposition in the induction of cell proliferation. Since deposited energy initiates events immediately, its removal, also supports the essentiality of the key event. Studies that do not deposit energy are observed to have no downstream effects. in vivo No energy deposition, as seen in the control of experiments of many studies does not lead to proliferating cells (Worgul et al., 1986; von Sallmann, 1952; Richards, 1966). Similarly, shielding 40 – 60% of the lens from X-irradiation shows a decreased response, starting 1-2 weeks after in vivo exposure to 1400 r X-rays (Pirie & Drance, 1959). 

Proliferation markers are also not incorporated into the cells at the same rate when no energy is deposited in cells. For example, within the lens transitional zone, 11 Edu+ cells/0.045mm2 were seen when exposed to 0.05 Gy X-rays, but only 5 EdU+cells/0.045 mm2 were detected in control groups. Similarly, the germinative zone of the lens displayed 28 Edu+cells/0.045 mm2 when exposed to 0.25 Gy, 4x the number EdU+ cells for control (Markiewicz et al., 2015). 

Cell numbers are not significantly increased when energy deposition does not occur. Control lens epithelium samples had 2.8x105 cells/ml, 0.8x the 3.5x105 cells/ml seen when exposed in vitro to low dose rate 0.01 Gy X-rays (Bahia et al., 2018). Lens epithelium cell area coverage reached 27 mm2 after 2 Gy of in vitro X-rays, but only 18 mm2 for control (Fujimichi & Hamada, 2014). 

• Exposure to radiation has been associated with the arrest of the cell cycle (Khan & Wang, 2022; Hein et al., 2014; Wang et al., 2018; Turesson et al., 2003). The cell cycle function is associated with the cell’s ability to undergo mitosis and generate additional cells (Khan & Wang, 2022; Reynolds & Schecker, 1995). Radiation turns on cell cycle checkpoints, causing cycle arrest (Wang et al., 2018; Turesson et al. 2003). When the cycle is arrested, cells are unable to progress to the next stage, meaning that any cells not in the mitotic phase would then be unable to proliferate (Hein et al., 2014; Khan & Wang, 2022). Several studies show doses as low as 10 mGy (of alpha particle irradiation on human fibroblast cells) leading to less proliferation than control groups (Khan & Wang, 2022). Other studies found that proliferation was either increased or decreased based on the time since irradiation. In the earlier stages, 4 to 7 days post-irradiation, there was a decrease in cell proliferation (von Sallmann et al., 1955; Barnard et al., 2022). During this time, larger radiation doses led to a larger decrease. After this point, cell proliferation began to increase and larger radiation doses led to increased proliferation (rabbits, 125, 250, 500, 1000, 2000 rep) (von Sallmann et al., 1955). Pirie and Drance also found a similar effect, but they noted a continued decrease in proliferation after the increase seen by von Sallmann et al. (1959).  

• Furthermore, LECs also see inconsistent results in radiation effects, with some radiation exposed cells forming colonies through excessive proliferation and others becoming inactivated or dead. This inactivation involves a long-term cell cycle arrest that is nonpermanent but does prevent proliferation from occurring (Fujimichi & Hamada, 2014). However, a subpopulation of LECs demonstrated increased sensitivity to radiation induced premature senescence and therefore, a cessation of proliferation for any cells not in mitosis (Hamada, 2017b). 

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