PubMed · 7442329
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R J Shmookler Reis, S Goldstein, C B Harley. 1980. Is cellular aging a stochastic process?. https://doi.org/10.1016/0047-6374(80)90081-0
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In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.
Human skin chronically exposed to UV light is known to accumulate iron and to have an increased ferritin content as compared to unexposed areas. Iron accumulation is also found in many inflammatory skin diseases. Cultured human fibroblasts loaded with iron by incubation with non-toxic concentrations of the ferric nitrilotriacetate complex have been irradiated with low (up to 15 J/cm2) and moderate (up to 45 J/cm2) UVA doses. At low irradiation doses, lipid peroxidation doubles without affecting the viability of iron-loaded cells. At higher irradiation doses (30 J/cm2) the photocytotoxicity of UVA towards iron-loaded cells increases in a concentration-dependent manner with the iron load. Thus, after exposure to 30 J/cm2 of UVA, the cytotoxicity is about 3-fold greater for cells incubated for 75 min with 100 microM of the ferric complex as compared to those not treated with the ferric complex. Incubation with desferrioxamine, an extremely efficient chelator of ferric ion or vitamin E, a radical scavenger which blocks the lipid peroxidation radical chain, leads to marked inhibition of the sensitizing effects of iron on lipid peroxidation but is less effective for the survival of cells exposed to UVA. A similar concentration-dependent protective effect of desferrioxamine was observed with cultured fibroblasts not treated with the ferric complex. It is suggested that the photoreduction of ferritin and/or other iron-containing proteins plays a significant role in the UVA-induced photocytotoxicity of skin fibroblasts.
When ML-1 human myeloid leukemia cells are exposed to DNA damaging agents, they exhibit dramatic changes in the expression of a variety of gene products. This includes an increase in p53 (wild-type), a decrease in BCL2, a p53-dependent increase in the BCL2 family member BAX, and increases in Growth Arrest and DNA Damage-inducible (GADD) genes such as GADD45; these changes occur as early events in a sequence that culminates in DNA damage-induced apoptosis. DNA damaging agents have now been tested for effects on expression of another BCL2 family member, MCL1, a gene expressed during ML-1 cell differentiation. Expression of MCL1 was found to increase upon exposure of ML-1 cells to various types of DNA damaging agents, including ionizing radiation, ultraviolet radiation, and alkylating drugs. The increase in MCL1 occurred rapidly and was transient, levels of the MCL1 mRNA being elevated within 4 h and having returned to near baseline within 24 h. An increase in the Mcl1 protein was also seen, with the maximal increase occurring at an intermediate dose of IR (5 Gray) and lesser increases occurring at either lower or higher doses. The increase in expression of MCL1 was further studied using a panel of human cell lines that includes cells containing or not containing alterations in p53 as well as cells sensitive or insensitive to the apoptosis-inducing effects of DNA damage. The DNA damage-induced increase in MCL1 mRNA did not depend upon p53 as it was seen in cells lacking functional p53. However, the increase did depend upon susceptibility to apoptosis as it was not seen in cells insensitive to apoptosis-induction by DNA damaging agents. These findings demonstrate that cytotoxic DNA damage causes an increase in the expression of MCL1 along with increases in GADD45 and BAX and a decrease in BCL2. Furthermore, while the increase in GADD45 is seen both in cells that undergo growth arrest and in cells that undergo apoptosis in response to DNA damage, alterations in the profile of expression of BCL2 family members occur exclusively in cells that undergo the apoptotic response, with some family members increasing through p53-dependent (BAX) and others through p53-independent (MCL1) pathways. Overall, expression MCL1 can increase during the induction of cell death as well as during the induction of differentiation.