A single low dose of Fe ions can cause long-term biological responses in NL20 human bronchial epithelial cells

Space radiation cancer risk may be a potential obstacle for long-duration spaceflight. Among all types of cancer space radiation may induce, lung cancer has been estimated to be the largest potential risk. Although previous animal study has shown that Fe ions, the most important contributor to the total dose equivalent of space radiation, induced a higher incidence of lung tumorigenesis per dose than X-rays, the underlying mechanisms at cellular level remained unclear. Therefore, in the present study, we investigated long-term biological changes in NL20 human bronchial epithelial cells after exposure to Fe ion or X-ray irradiation. We found that compared with sham control, the progeny of NL20 cells irradiated with 0.1 Gy of Fe ions showed slightly increased micronucleus formation, significantly decreased cell proliferation, disturbed cell cycle distribution, and obviously elevated intracellular ROS levels accompanied by reduced SOD1 and SOD2 expression, but the progeny of NL20 cells irradiated with 0.9 Gy of X-rays did not show any significant changes. More importantly, Fe ion exposure caused much greater soft-agar colony formation than X-rays did in the progeny of irradiated NL20 cells, clearly suggesting higher cell transformation potential of Fe ions compared with X-rays. These data may shed the light on the potential lung tumorigenesis risk from Fe ion exposure. In addition, ATM inhibition by Ku55933 reversed some of the changes in the progeny of Fe ion-irradiated cells but not others such as soft-agar colony formation, suggesting complex processes from DNA damage to carcinogenesis. These data indicate that even a single low dose of Fe ions can induce long-term biological responses such as cell transformation, etc., suggesting unignorable health risk from space radiation to astronauts.     

Computerized video time-lapse analysis of apoptosis of REC:Myc cells X-irradiated in different phases of the cell cycle

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20-60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G(1) phase divided one to six times and survived 40-120 h before undergoing apoptosis compared to only one to two times and 5-40 h for cells irradiated in G(2) phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.     

Computerized video time-lapse (CVTL) analysis of cell death kinetics in human bladder carcinoma cells (EJ30) X-irradiated in different phases of the cell cycle

The purpose of this study was to quantify the modes and kinetics of cell death for EJ30 human bladder carcinoma cells irradiated in different phases of the cell cycle. Asynchronous human bladder carcinoma cells were observed in multiple fields by computerized video time-lapse (CVTL) microscopy for one to two cell divisions before irradiation (6 Gy) and for 6-11 days afterward. By analyzing time-lapse movies collected from these fields, pedigrees were constructed showing the behaviors of 231 cells irradiated in different phases of the cell cycle (i.e. at different times after mitosis). A total of 219 irradiated cells were determined to be non-colony-forming over the time spans of the experiments. In these nonclonogenic pedigrees, cells died primarily by necrosis either without entering mitosis or over 1 to 10 postirradiation generations. A total of 105 giant cells developed from the irradiated cells or their progeny, and 30% (31/105) divided successfully. Most nonclonogenic cells irradiated in mid-S phase (9-12 h after mitosis) died by the second generation, while those irradiated either before or after this short period in mid-S phase had cell deaths occurring over one to nine postirradiation generations. The nonclonogenic cells irradiated in mid-S phase also experienced the longest average delay before their first division. Clonogenic cells (11/12 cells) divided sooner after irradiation than the average nonclonogenic cells derived from the same phase of the cell cycle. The early death and long division delay observed for nonclonogenic cells irradiated in mid-S phase could possibly result from an increase in damage induced during the transition from the replication of euchromatin to the replication of heterochromatin.     

Persistent expression of morphological abnormalities in the distant progeny of irradiated cells

The phenomenon of delayed heritable lethal damage (often referred to as ``lethal mutations') in the progeny of cells which survive irradiation is now well established, but little is known of the mechanism by which this cell death occurs. Current theories suggest a generalised genomic instability affecting all cells which leads to the production of some mutations which are lethal, or alternatively that a lethal mutation gene is activated, mutated or induced by radiation and leads to persistent and random cell death at high levels in the progeny. The aim of this study was to look at the morphology of progeny of irradiated cells at various times after irradiation to establish how widespread morphological abnormalities were in the population and whether there was any evidence that such abnormalities were clonal. Using two different cell lines, the results showed that morphological evidence possibly suggestive of apoptosis occurred in the cultures after all doses of radiation and up to 45 cell doublings after exposure. There was no evidence of a decrease in the numbers of damaged or dead cells in colonies with number of divisions after irradiation, or with decreasing original radiation dose. There was a significant dose-dependent increase in the number of cells with microvilli for both cell lines. The dose-dependency of this effect did not change with number of divisions after irradiation. It is clear that morphological evidence of cellular damage persists for several generations after the initial exposure. The effects are widespread in the cell population, and their constancy over time argues strongly for a general instability and against a clonal mechanism, since clonal descendants should die out and leave undamaged survivors. The lack of evidence for necrosis or senescence together with many morphological changes in the cultures suggestive of apoptosis could indicate an active mechanism of cell death. It is concluded that survivor populations of irradiated cells from two widely different mammalian cell lines demonstrate an altered phenotype including gross morphological changes. These result in a higher probability that cell division will fail to yield two healthy progeny. Received: 22 January 1996 / Accepted in revised form: 24 September 1996     

Asymmetric somatic cell hybridization in plants

Summary An investigation into the possible application of UV radiation as a pretreatment for the donor cells in asymmetric plant cell hybridization protocols has been carried out. A comparison was made between the effects of UV doses in the range 700–4200 J/m² and those of ⁶⁰Co gamma radiation over the range 0.15–1 kGy on Beta vulgaris suspension cell protoplasts. The investigation had two aspects. Firstly, alterations to cell physiology (cell wall resynthesis, viability, division and colony formation) in irradiated protoplasts were examined during a 4-week culture period. Results have indicated that a dose of 700 J/m² UV is necessary to prevent further cell division and colony formation in these cells. A dose of 0.15 kGy gamma radiation generally prevented colony formation, although some early cell division did occur (as was also observed even after 0.45 kGy had been applied). Membrane integrity, as measured after 6 days, using fluorescein diacetate staining, was not affected by either treatment within the dose ranges applied. Secondly, denaturing (alkaline) gel electrophoresis, in association with a pulsed field gel DNA preparation technique, was used to determine the degree of in vivo DNA damage following the radiation treatments. After UV radiation, considerable fragmentation of the DNA was observed, the extent of which was dose-dependent. Gamma radiation, however, appeared to result in fewer DNA lesions, with only the 1 kGy treatment revealing a pattern significantly altered from that of the control. These results augur well for the potential use of UV radiation in asymmetric fusion experiments.     

Repair of radiation-induced damage to the cell division mechanism of Escherichia coli

Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), William D. Fisher, Alice A. Hardigree, and George E. Stapleton. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli. J. Bacteriol. 91:737-742. 1966.-Microscopic observations of irradiated populations of filamentous Escherichia coli cells indicated that filaments can be induced to divide by a substance donated by neighboring cells. We have made this observation the basis for a quantitative technique in which filaments are incubated in the presence of nongrowing donor cells. The presence of "donor" organisms promotes division and subsequent colony formation in filaments. "Donor" bacteria do not affect nonfilamentous cells. An extract of "donor" cells retains the division-promoting activity. The extract has been partially fractionated, and consists of a heat-stable and a heat-labile component. The heat-stable component is inactive in promoting cell division, but enhances the activity of the heat-labile component. The division-promoting system is discussed as a radiation repair mechanism and as a normal component of the cell division system in E. coli.     

Clonal heterogeneity in delayed decrease of plating efficiency of irradiated HeLa cells

The clonogenic potential of progeny of irradiated HeLa cells was studied at different times after single doses of 4–12 Gy. The dose-dependent decrease in plating efficiency that was observed resembled the effect termed delayed lethal mutation by Seymour et al. (1986). The effect decreased with time after irradiation. Individual clones of irradiated and non-irradiated cells were isolated, expanded and replated 5 weeks after irradiation, i.e., after between 200000 and 1000 000 progeny had formed from the individual parent cell. The plating efficiency of progeny of unirradiated cells did not vary much, whereas clonal progeny of irradiated cells had plating efficiencies ranging from 3% to 76%. The plating efficiency was not related to the cell number in the original clone.     

Quantitative measurement of damage caused by 1064-nm wavelength optical trapping of Escherichia coli cells using on-chip single cell cultivation system

We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E. coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive.     

Effect of starving and dowex 50 treatment on growth of normal and x-irradiated yeast

1. Effects of starvation or treatment with a cation exchange resin, dowex 50, parallel in some respects those seen earlier on the respiration and fermentation of bakers' yeast receiving 90,000 r of 250 kv. x-rays. Starvation increased the radiosensitivity of cell division processes whether measured by colony formation or by turbidimetric determination of growth in a liquid medium. The dowex 50 enhanced the radiation effect by the latter measure but appeared to increase colony formation of irradiated yeast. 2. The effects on growth differ from those on respiration and fermentation in that the exchange resin treatment did not inhibit colony formation further, and neither starvation nor resin appreciably altered the growth of non-irradiated yeast. 3. Two effects of radiation are seen in these experiments: (a) a permanent inhibition of growth, and (b) a temporary inhibition of the remaining cells resulting in delay of growth. 4. The irradiated cell is more dependent on certain aspects of its environment in terms of growth responses as well as in terms of metabolism (i.e. respiration and fermentation). Whether or not potassium plays a role in the growth response as it does in the metabolic response cannot be ascertained from the present data.     

MITOSIS, CELL WALL AND FLAGELLA SYNTHESIS IN SYNCHRONIZED CULTURES OF THE PRASINOPHYTE, SCHERFFELIA DUBIA

Cell division occurs within the parental cell wall, yielding two progeny cells. Since Scherffelia dubia sheds all four flagella prior to cell division, the maturing progeny cells must regenerate new cell walls and flagella during and/or after cytokinesis. To better understand these processes, we have synchronized cell division in cultures of S. dubia and observed all stages of mitosis, cytokinesis, and progeny cell maturation, including flagella and cell wall formation, via DAPI staining of fixed cells, DIC microscopy of live cells embedded in agarose and standard TEM. Microscopical observations revealed the following sequence of events: 1) Golgi stacks divide during late interphase and immediately begin producing theca scales; 2) deflagellation and release of the parental cell wall from the plasma membrane occurs during early prophase; 3) synthesis of theca and flagella scales within the Golgi and/or scale reticulum continues throughout mitosis; 4) during cytokinesis, a coalescence of vesicles containing theca scales at the posterior end of the cell results in a cleavage furrow slightly diagonal to the cells' longitudinal axis (40 min); 5) post-mitotic nascent basal body formation and flagella elongation at the inherited basal bodies (and later at the mature nascent basal bodies) occurs concurrently with continued cell wall synthesis; 6) the cleavage furrow rotates into a transverse position (35 min); 7) reorientation of the nuclei results in a "head to tail" orientation of the maturing progeny cells; and 8) matured progeny cells emerge from the posterior end of the parental theca not before 8 hrs after the onset of mitosis.     

Potentiation by caffeine of ultraviolet-light damage in cultured human cells.

Five cultured human cell lines (T-1 kidney, Chang liver, H.Ep. No. 2, HeLa-S3 and HeLa-O) were irradiated with ultraviolet light and immediately exposed to 1.0 and 3.0 mM caffeine for 44 h thereafter. This caffeine treatment reduced the surviving fraction (assayed by colony formation) of the irradiated population, but did not significantly reduce the colony-forming ability of unirradiated control cells. These findings suggest that many cultured human cell lines exhibit post-UV potentiation of potentially lethal damage by caffeine.     

Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation

Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation, a process known as the bystander effect. Bystander effects can also lead to chromosomal/genomic instability within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The factors that mediate this cellular communication can be transferred between cells via gap junctions or released into the extracellular media following irradiation, but their nature has not been fully characterized. In this study we tested the hypothesis that the bystander effect mediator contains an RNA molecule that may be carried by exosomes. MCF7 cells were irradiated with 2 Gy of X rays and the extracellular media was harvested. RNase treatment abrogated the ability of the media to induce early and late chromosomal damage in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the bystander effect, and genomic instability, are at least in part mediated by exosomes and implicate a role for RNA.     

Isolation and characterization of highly radioresistant malignant hamster fibroblasts that survive acute gamma irradiation with 20 Gy

To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK-) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK- cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D0 for PIC-20 cells was 7.4 +/- 0.2 Gy, which is three times higher than that for parental cells (2.5 +/- 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK- cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK- cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.     

Cell growth and division in Escherichia coli: a common genetic control involved in cell division and minicell formation

Escherichia coli Div 124(ts) is a conditional-lethal cell division mutant formed from a cross between a mutant that produces polar anucleated minicells and a temperature-sensitive cell division mutant affected in a stage of cross-wall synthesis. Under permissive growth temperature (30 C), Div 124(ts) grows and produces normal progeny cells and anucleated minicells from its polar ends. When transferred to nonpermissive growth temperature (42 C), growth and macromolecular synthesis continue, but cell division and minicell formation are inhibited. Growth at 42 C results in formation of filamentous cells showing some constrictions along the length of the filaments. Return of the filaments from 42 to 30 C results in cell division and minicell formation in association with the constrictions and other areas along the length of the filaments. This gives rise to a "necklace-type" array of cells and minicells. Recovery of cell division is observed after a lag and is followed by a burst in cell division and finally by a return to the normal growth characteristic of 30 C cultures. Recovery of cell division takes place in the presence of chloramphenicol or nalidixic acid when these are added at the time of shift from 42 to 30 C, and indicates that a division potential for filament fragmentation is accumulated while the cells are at 42 C. This division potential is used for the production of both minicells and cells of normal length. The conditional-lethal temperature sensitive mutation controls a step(s) in cross-wall synthesis common to cell division and minicell formation.     

Transient erythropoietic spleen colonies: effects of erythropoietin in normal and genetically anemic W/Wv mice.

Properties of the cells (TE-CFU) that give rise within four to six days to transient endogenous erythropoietic spleen colonies in irradiated mice have been investigated. The results obtained indicate that (1) erythropoietic maturation within such colonies is highly erythropoietin-dependent, (2) the population size of TE-CFU is not erythropoietin-dependent, (3) initial exposure to a high dose of erythropoietin followed by continuing exposure to lower doses is required for maximal efficiency of colony formation by TE-CFU, (4) successful transplantation of TE-CFU has not been achieved, but they appear among the progeny of transplanted hemopoietic cells, (5) TE-CFU are defective in mice of genotype W/Wv. These findings are consistent with the view that the TE-CFU assay detects a class of early erythropoietin-sensitive progenitor cells committed to erythropoietic diffferentiation, rather than "abortive" colony formation by pluripotent stem cells.     

Clonal analysis of progenitor cell commitment to granulocyte or macrophage production

Granulocyte-macrophage colony formation by C57BL bone marrow cells was initiated in agar cultures either by the granulocyte-macrophage stimulus, GM-CSF, or by the predominantly macrophage stimulus, M-CSF. After 24 hours, paired daughter cells of granulocyte-macrophage colony-forming cells (GM-CFC) were separated by micromanipulation and one cultured in GM-CSF, the other in M-CSF. From the differentiation pattern of the resulting colonies, irreversible commitment of some cells occurred during the first 24 hours and completion of the first cell division. A similar result was obtained using granddaughter cells present after 24 hours of incubation. However, when intact developing day 2 and days 3 clones were cross-transferred to GM-CSF or M-CSF recipient cultures, irreversible commitment was more obvious. Most M-CSF-initiated clones exhibited irreversible commitment to macrophage formation in GM-CSF cultures and a high proportion of GM-CSF-initiated clones continued to produce granulocyte progeny after transfer to M-CSF. The results indicated that GM-CSF and M-CSF can irreversibly commit the progeny of GM-CFC respectively to granulocyte or macrophage production. While for some GM-CFC this occurs within 24 hours and one cell division, for many cells, the process is slower and requires an incubation period of up to 48 hours and/or several cell divisions. Calculations from the data indicated that two-thirds of GM-CFC in adult C57BL marrow are biresponsive and respond to stimulation both by GM-CSF and M-CSF.     

All colonies of CHO-K1 cells surviving gamma-irradiation contain non-viable cells.

This paper addresses the problem of the production of defective cells within clones arising from irradiated progenitor cells and is specifically aimed at answering the question of whether lethal mutations result from a generalised effect which lowers the ability of all the progeny to divide successfully or whether it represents a late expressed but unique lethal defect induced by radiation which occurs in some cells only and which causes those cells only to cease dividing. The results obtained from autoradiographic analysis of cells within individual surviving colonies (i.e. containing more than 150 cells) suggests that some cells in all clones are not synthesizing DNA over a 9-h period and that the proportion of non-synthesising cells rises with increasing dose of radiation from less than 3% in the controls to 80-85% after a progenitor dose of 12.5 Gy. Because of the possibility that cells had longer division times post irradiation, these results were repeated using Ki67 antibody labelling, a technique which identifies cells which are in cycle. The results were similar. This suggests the non-labelled cells were not reproducing. Both techniques were also used to look at the % labelling of morphologically abnormal cells in the colonies. The results suggested that up to 35% of these abnormal cells were actively cycling and about 20% were synthesising DNA. Abnormal cells did not appear in subcultures of survivor progeny suggesting that they may have failed to replate successfully and may contribute to the lethally mutated population. The idea that radiation induces a general instability in the cell population was supported by experiments where growth and the plating efficiency of irradiated progeny was measured daily. This revealed that the growth curves deviated from the control by a constant factor suggesting a division probability of about 70% of the control level after a progenitor dose of 10 Gy. The results are discussed in the context of their significance for survival curve analysis and for radiotherapy and radiation protection results.     

Somatic segregation and rate of greening after ultraviolet irradiation of Euglena gracilis.

1. During multiplication of irradiated cells, a segregation may take place between bleached cells, whose progeny is unable to green, and green ones. Some of the green cells give progenies exclusively made of green cells; the progeny of others is partly composed of bleached cells. 2. If one assumes that greening results from the activity of functional units endowed with genetic continuity (Plastidial Segregating Units = PSU), segregation of these units seems to occur according to a model involving random sorting out during the three first divisions. During the following divisions, functional units seem to multiply faster than those impaired by irradiation. 3. The greening rate of colonies issued from irradiated cells seems to be conditioned mostly by the number of functional PSU remaining in the mother cell of the colony.     

The dependence of the formation of stromal CFU-f colonies on the stimulating action of hematopoietic cells

CFU-f-derived stromal colony formation was accomplished in adherent marrow cell cultures (AMCC) with serum-rich medium. It turned out to require additional stimulation by hemopoietic feeder cells: by irradiated marrow cells and spleen cells if they possess megakaryocytes and platelets or by platelets from the blood. PDGF, EGF and IL-3 did not substitute the colony stimulating activity of feeder cells. Thymus, lymph node cells and blood leucocytes had no colony stimulating activity. At low oxygen concentrations which improve colony formation the stimulating activity of hemopoietic feeder cells was expressed, as well. Thus, CFU-f colony formation depends on stimulation by hemopoietic cells in addition to serum growth factors. In full populations of marrow cells the CFU-f colony formation is stimulated by marrow cells which accompany the CFU-f.     

Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G₂/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.