Immortal phenotype of the HeLa variant D98 is recessive in hybrids formed with normal human fibroblasts

Normal human cells such as human diploid fibroblasts (HDF) have a finite proliferative lifespan in culture. Previous studies have shown that the limited lifespan phenotype is dominant in cell hybrids formed by fusion of HDF to at least 23 different kinds of immortal human cells. However, two independent studies reported that hybrid clones formed by the fusion of HDF to the HeLa variant D98 had unlimited division potential. Those results were potentially very important because they implied that a) there is a dominant mechanism for immortalization of human cells in addition to the well-documented recessive mechanism, and b) a dominant mechanism would lend itself to identification of the immortalizing gene. Consequently, we carried out more detailed studies of the behavior of D98 cells in hybrids. Our results indicate that the majority of D98 x HDF hybrid clones exhibit a clear-cut finite proliferative lifespan phenotype. In addition, these hybrid cell populations often give rise to an immortal focus of cells that can be seen to take over the population of mortal cells at the end of their lifespan. This phenomenon reconciles our data with the previous reports of immortal D98 x HDF hybrid clones and leads us to conclude that D98 cells do not express a dominant immortalizing gene.     

Ultraviolet enhanced reactivation of a human virus: effect of delayed infection

The ability of UV-irradiated herpes simplex virus to form plaques was examined in monolayers of CV-1 monkey kidney cells preexposed to UV radiation at different intervals before virus assay. From analysis of UV reactivation (Weigle reactivation) curves it was found that as the interval between cell UV irradiation (0-20 J/m2) and initiation of the virus assay was increased over a period of five days, (1) the capacity of the cells to support unirradiated virus plaque formation, which was decreased immediately following UV exposure to the monolayers, increased and returned to approximately normal levels within five days, and (2) at five days an exponential increase was observed in the relative plaque formation of irradiated virus as a function of UV fluence to the monolayers. For high UV fluence (20 J/m2) to the cells, the relative plaque formation by the UV-irradiated virus at five days was about 10-fold higher than that obtained from assay on unirradiated cells. This enhancement in plaque formation is interpreted as a delayed expression of Weigle reactivation. The amount of enhancement resulting from this delayed reactivation was several fold greater than that produced by the Weigle reactivation which occurred when irradiated herpes virus was assayed immediately following cell irradiation.     

Experimental radiation pneumonitis. Corticosteroids increase the replicative activity of alveolar type 2 cells

Corticosteroid administration during radiation pneumonitis in mice markedly improves the physiologic abnormalities and decreases mortality, an effect that has been attributed to the stimulation of surfactant synthesis and secretion by type 2 alveolar epithelial cells. In the present experiments we explored the effects of corticosteroids on the replicative activity of type 2 cells of lethally irradiated lungs at the height of the radiation reaction. The labeling index of type 2 cells of irradiated mice was increased threefold above that of sham-irradiated controls. Corticosteroids given continuously from 10 weeks after thoracic irradiation further increased the type 2 cell labeling index another threefold above that of irradiated untreated mice. The enhanced reproductive activity of type 2 cells following thoracic irradiation is seen as a protective response that is augmented by corticosteroids, whose effect may be both to improve the physiology of the alveolar surface and to maintain the population of alveolar epithelial cells. The bearing of this result on the controversial role of the type 2 cell as a target in radiation pneumonitis is discussed.     

The effect of UV irradiation on proliferation and life span of human diploid fibroblast-like cells

Summary The effect of low dose UV irradiation on the reinitiation of proliferative activity and on the life span of human diploid fibroblast-like cells is described. Cells were exposed to UV at confluence or after maintenance in an arrested state. Cell division was stimulated immediately after UV irradiation or after an additional post-UV incubation period. Arrested populations of all in vitro ages exhibited a greater sensitivity to UV and the reinitiation of proliferation was enhanced by post-UV incubation before stimulation. Ultraviolet light had no effect on life span regardless of in vitro cell age, culture state at the time of exposure, or the presence of a postirradiation period of arrest.     

Effects of 50 Hz magnetic field on cell cycle kinetics and the colony forming ability of budding yeast exposed to ultraviolet radiation.

To investigate the effects of extremely low frequency magnetic fields on ultraviolet radiation (UV) exposed budding yeast, haploid yeast (Saccharomyces cerevisiae) cells of the strain SEy2101a were exposed to 50 Hz sine wave magnetic field (MF) of 120 microT with simultaneous exposure to UV radiation. Most of the UV energy was in the UVB range (280-320 nm). The biologically weighted (CIE action spectrum) dose level for the UV radiation was 175 J/m2. We examined whether 50 Hz MF affected the ability of UV irradiated yeast cells to form colonies (Colony Forming Units, CFUs). In addition, the effect of coexposure on cell cycle kinetics was investigated. Although the significant effect of MF on the cell cycle phases of UV exposed yeast cells was seen only at one time point, the overall results showed that MF exposure may influence the cell cycle kinetics at the first cycle after UV irradiation. The effect of our particular MF exposure on the colony forming ability of the UV irradiated yeast cells was statistically significant 420 min after UV irradiation. Moreover, at 240, 360, and 420 min after UV irradiation, there were fewer CFUs in every experiment in (UV+MF) exposed populations than in only UV exposed yeast populations. These results could indicate that MF exposure in conjunction with UV may have some effects on yeast cell survival or growth.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0–30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells

Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.     

Radiotoxicity of incorporated [3H]thymidine Consequences for the interpretation of FLM data of irradiated cell populations

Abstract. Problems in the interpretation of FLM data of externally irradiated cell populations are mainly due to the interference of radiation effects with radiotoxic effects originating from incorporated [³H]thymidine. These problems were investigated using L-929 cells flash labelled in vitro with [³H]thymidine (30 min, 0.3 μCi/ml, 40 Ci/mM) and irradiated with 2 Gy of 200 kV X-rays; the fractions of labelled mitoses and the index of labelled and of unlabelled mitoses were determined. The results showed that the FLM is not an adequate parameter to quantify the early cell kinetic changes in irradiated cell populations.     

Cell cycle changes and cytotoxicity in irradiated cultures of bovine aortic endothelial cells

The purpose of this experiment was to determine the effect of ionizing radiation on cell number, lactate dehydrogenase (LDH) release, cell cycle distribution, [3H]thymidine incorporation, and autoradiographic labeling index in bovine aortic endothelial cells in vitro. Confluent endothelial monolayers were exposed to single doses of 0.5-10 Gy of 60Co gamma rays and were analyzed from 2 to 24 h postirradiation. Irradiated monolayers exhibited a time- and dose-dependent decrease in cell number, increase in LDH release, and redistribution of cells in the cell cycle. Cell cycle redistribution included an increase in the proportion of cells in S phase at 4 h after irradiation and a decrease in S phase at 24 h. The cells also exhibited a decrease in [3H]thymidine incorporation as early as 2 h after 5 Gy. This represented the most rapid radiation response observed in the present study. These data demonstrate that radiation cytotoxicity in confluent, plateau-phase endothelial monolayers is accompanied by changes in the cell cycle distribution of adherent cells, and that reduced [3H]thymidine incorporation is an early marker of radiation injury in this clinically important cell type.     

Purified scrapie prions resist inactivation by UV irradiation.

The development of effective purification protocols has permitted evaluation of the resistance of isolated scrapie prions to inactivation by UV irradiation at 254 nm. Prions were irradiated on ice with doses of UV light ranging up to 120,000 J/m2. UV dosimetry experiments, performed with Saccharomyces cerevisiae plasmid DNA or eucaryotic cells, indicated that under these experimental conditions an incident UV dose of 10 J/m2 formed 2 thymine dimers per 5.1 X 10(6) daltons of eucaryotic cell DNA. The D37 values for scrapie prions ranged from 17,000 to 22,000 J/m2; D37 values were also determined for virus, viroid, and enzyme controls. The number of pyrimidine dimers formed was correlated with the D37 values obtained for irradiated prions and target nucleic acids. The D37 value for bacteriophage M13, 6.5 J/m2, occurred at a dose that would form 0.56 dimers per target genome; the D37 for potato spindle tuber viroid, 4,800 J/m2, occurred at a dose that would form about 24 dimers per target viroid. The D37 value for an EcoRI restriction site, a target of 12 bases, occurred at a dose that would correspond to the formation of 0.89 thymine dimers per target site. The D37 value for prions occurred at a dose that would form 1 dimer in every 4 bases of single-stranded target nucleic acid. If the putative scrapie nucleic acid were double-stranded and readily repairable after UV damage, then the prion D37 value could reflect a nucleic acid molecule of 30 to 45 base pairs. While the D37 value for prions fell within the range of pure protein targets, our experiments cannot eliminate the possibility that a prion contains a small, highly protected nucleic acid molecule.     

Effect of the distance between adherent mesenchymal stem cell and the focus of irradiation of femtosecond laser on cell replication capacity

The effect of femtosecond laser irradiation on adherent mesenchymal stem cell was investigated with the aim to develop a novel noninvasive cell purification system. A single mesenchymal stem cell was irradiated with a femtosecond laser on the center of the cell using several energy values and the cell lost its replication capacity with one time irradiation with an energy of 2.0 μJ. Besides at a neighbor point in the major axis, when irradiated in the minor axis at a distance shorter than 10 μm, the cell stopped its replication capacity. The accumulation effect of cell damage caused by multiple laser shots at a neighbor point in the minor axis was correlated with the critical distance at which the cell lost its replication capacity. Finally, a novel equation of laser cell damage as a function of laser pulse energy and laser shot number is proposed.     

Ultraviolet radiation response to the physical properties of the reconstituted nucleohistone

The effect of UV irradiation on the reconstituted nucleohistone have been studied with reference to its (nucleohistone) changes in physical properties, after irradiation at different UV doses. The rate of fall of specific viscosity ratio of the reconstituted nucleohistone as a function of UV dose decreased gradually with the increasing histone to DNA weight ratio (r). This effect, was not observed when the histones remained dissociated from DNA, in high ionic strength (1.5 M NaCl). Histone-DNA complex (r=0.97) irradiated up to a dose of 3.6×10⁴ J/m² had a stable melting temperature unlike free DNA where UV irradiation lowered the melting temperature and the heterogeneous melting profiles were observed. Rate of formaldehyde reaction, with DNA recovered from the irradiated complex, was slower than that with native DNA treated at the same dose. All this suggested that the effect of UV in the DNA of the nucleohistone was less, compared to that in free DNA.     

Proliferation and tumorigenity of murine hepatoma cells irradiated with polychromatic visible and infrared light

In experiments in vitro, the effects of polychromatic visible (VIS) light combined with polychromatic infrared light (VIS-IR, 480–3400 nm) and the effects of the entire spectrum of VIS radiation (385–750 nm) on viability and proliferative activity of the murine hepatoma cells MH22a were studied. In experiments in vivo, changes in the tumorigenic properties of cells MH22a were studied after the same kinds of light exposure. It was shown that irradiation of hepatoma cells with two kinds of polychromatic light at a wide range of doses (4.8–38.4 J/cm²) did not lead to an increase in the number of dead cells for 24–72 h of cultivation and did not cause deceleration of the hepatoma cell proliferation; moreover, the VIS-IR light at a dose of 4.8 J/cm² and the VIS light at a dose 38.4 J/cm² even promoted more intense cell proliferation after 24 h. In cells irradiated with VIS-IR and VIS light, the proliferation index rose by 1.6 and 1.4 times, respectively, and the time of the cells’ number doubling decreased as compared with control. Studying the tumorigenic properties of irradiated tumor cells has shown that, for 30 days after transplantation to syngenic mice C3HA of hepatoma cells 24 h after their irradiation with VIS-IR light at a dose of 4.8 J/cm², the tumor volume decreased significantly (2.6–4.1 times) at all periods of observation, while the incidence of tumor formation decreased, whereas the survival of the tumor-bearing mice did not change. Transplantation of cells irradiated with the same light at a dose of 9.6 J/cm² did not lead to significant changes in the tumor volume, the tumor formation incidence, and animal survival. The main contribution to the antitumor effect of VIS-IR light seems to be made by the VIS component, as transplantation into mice of cells irradiated with VIS light alone at a dose of 38.4 J/cm² also stimulating proliferation of hepatoma cells in vitro resulted in a decrease of their tumorigenic properties. However, the IR component in the combined VIS-IR radiation enhanced the antitumor effect of the VIS light; as a result, it was manifested after use of doses eight times lower (4.8 J/cm²) than in the case of VIS light alone (38.4 J/cm²). Mechanisms of the decrease of tumorigenic properties of hepatoma cells after irradiation with polychromatic light at doses stimulating their proliferation in vitro are studied.     

Growth-stimulating effect of UF-irradiated blood. I. The radiation dose dependence of the initial growth potencies of blood and of the functional state of the target cells

UV irradiation (UVI) of donor blood in the apparatus used in hospitals of the USSR with the therapeutic aim of autotransfusion of UV-irradiated blood (AUVIB), results in an increase of connective tissue cell growth potency: being added into culture media the supernatants of irradiated blood stimulate DNA-synthetic and proliferative activity of cultured human embryonic cells. The high activity of cells persists for about 2 days. The effect is great with low initial levels of cell proliferative activity. In this case the effect is maximum (about 125% of the control). It is suggested that the above effect may be involved in the mechanism of stimulation of regeneration processes in the organism after AUVIB.     

Effects of recombinant human macrophage colony-stimulating factor on proliferation, differentiation and survival of Kupffer cells in the liver of adult mice

OBJECTIVE: To investigate the effects of macrophage colony-stimulating factor (M-CSF) on the proliferation, differentiation and survival of Kupffer cells in the liver of adult mice. STUDY DESIGN: By the combined method of autoradiography with [3H]thymidine and immunohistochemistry using a monoclonal antibody against mouse macrophages (F4/80 or BM8), the labeling rate of [3H]thymidine in macrophages within the liver sinusoids was examined at various intervals after single flash labeling with [3H]thymidine in adult mice with or without daily administration of recombinant human M-CSF. RESULTS: A minor population of Kupffer cells (about 2%) possessed proliferative capacity under a normal steady state condition. With time after flash labeling, the influx of monocytes and their differentiation into macrophages were demonstrated in the liver, and their labeling rate returned to the baseline level one week later. Afterward, the labeling rate of Kupffer cells was maintained at the baseline level until the end of five weeks. Administration of M-CSF enhanced the proliferative capacity of Kupffer cells, increased the number of macrophages and delayed the time of peaking. However, it did not prolong the survival of Kupffer cells. CONCLUSION: In normal mice, Kupffer cells can survive for at least five weeks. Daily M-CSF administration induces the increased number and proliferative capacity of Kupffer cells.     

Proliferative response of bystander cells adjacent to cells with incorporated radioactivity.

BACKGROUND: In a recent study, we showed that cells irradiated with gamma-rays stimulate cell growth of unirradiated (bystander) cells, when the two populations are co-cultured as a mixture. Direct cell-to-cell contact appears to be a prerequisite for the proliferative response of the bystander cells. The aim of the current work is to investigate the possible proliferative bystander effects caused by intracellular irradiation with incorporated radionuclides, specifically the short-range beta particle emitter, tritium ((3)H). METHODS: Subconfluent monolayers of rat liver epithelial cells (WB-F344) were incubated in the presence of (methyl-(3)H)thymidine ((3)HTdR) at concentrations ranging between 5.2 kBq/ml and 57.8 kBq/ml for 18 h. Radiolabeled cells, containing between 0.7 x 10(-3) Bq/cell and 8.8 x 10(-3) Bq/cell were mixed with unlabeled (i.e., bystander) cells in a ratio of 1:1 and cultured together for 24 h followed by an flow cytometry (FCM) study of their proliferation. In order to discriminate the two populations of co-cultured cells, one cell population (unlabeled bystander cells) was stained with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE), which metabolizes intracellularly. The absorbed doses received by the radiolabeled cells that contained 0.7 x 10(-3), 2.5 x 10(-3), and 8.8 x 10(-3) Bq/cell were 0.14, 0.49, and 1.7 Gy, respectively. RESULTS: Cells that were not treated with tritiated thymidine (unlabeled cells), in the presence of radiolabeled cells that received absorbed doses from 0.14-1.7 Gy, showed enhanced cell growth by approximately 9 to 10%. CONCLUSIONS: Cells labeled with (3)HTdR can induce increased proliferation in neighboring unlabeled bystander cells. FCM provides an excellent basis for characterization of proliferative bystander effects in co-culture systems.     

Effect of foreign bodies on the gallbladder mucosa of guinea pigs

Foreign bodies were introduced in the gallbladder of the guinea pig in order to study their effect on the cell proliferative parameters in the gallbladder mucosa. In vitro labeling with radioactive thymidine and autoradiographies were used. Our data show that the surgical manipulation of the control gallbladders had no effect on the mucosa. The introduction of polystyrene bodies was accompanied by a significant increase in DNA synthesis activity in the gallbladder epithelium.     

5-Azacytidine-induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts.

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.     

Effect of glutathione on UV induction of prophage lambda

The effect of glutathione (GSH) on the ultraviolet (UV) induction of lambda prophage was investigated in lysogenic Escherichia coli. The data showed that extracellular GSH could inhibit the UV induction of lambda prophage. The inhibitory rates were concentration dependent, and the maximal rate obtained was 94% with 3.0 M GSH. The effect was also measured in three different lambda lysogens: a wild-type strain (wt), an isogenic GSH-deficient strain, and an isogenic strain producing increased amounts of GSH. The result showed that when subjected to UV irradiation (254 nm, 60 J m⁻²), GSH-deficient strain was approximately fivefold more sensitive to be lysed than wt, whereas the strain with higher intracellular GSH levels was only 28% susceptible to be lysed. With electron spin resonance and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated lysogenic cells and the intensity of signals was influenced by GSH levels. These results indicate that GSH can significantly inhibit the UV induction of lambda prophage, and that this effect is correlated to its capacity to scavenge free radicals generated after UV irradiation.