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Our interest in cellular stress resistance emerged from the finding that fibroblasts derived from young adult Snell dwarf mice were resistant to multiple forms of cytotoxic stress.
Stress resistance in mouse primary fibroblast cultures
Mutations in worms and flies that lead to increased life span very often also endow these invertebrates with high levels of resistance to several forms of cellular stress, including ultraviolet irradiation, heat exposure, and oxidation stress. To see if mutations that increase mouse longevity also lead to cellular stress resistance, we evaluated stress resistance in vitro of fibroblast cell lines derived from the skin of Snell dwarf mice, in which a mutation at the Pit-1 gene leads to endocrine defects, small body size, and a 40% increase in mean and maximal life span. The results showed [PubMed] that fibroblasts from dwarf mice were resistant to cell death induced by each of five different stress agents: heat, UV light, hydrogen peroxide, paraquat, and the heavy metal cadmium. This finding has several implications. First, it suggests that the long life span and exceptional disease resistance of the dwarf mice may be due to an intrinsic resistance, at the cellular level, to multiple forms of stress. Second, it implies that stress resistance of fibroblasts, and perhaps also other cell types, can be modulated by early life exposure to varying levels of hormones including IGF-I and thyroid hormones, and that the mechanism of resistance is epigenetic, i.e. related to alterations in gene expression patterns that survive multiple rounds of mitosis in vitro. Third, it suggests that the relationships among hormonal patterns, organismic longevity and cell stress resistance may have emerged very early in eukaryote evolution, i.e. prior to the divergence between flies, worms, and mammals.
Follow-up studies have added useful detail to this initial picture. (1) Similar stress resistance profiles were seen using fibroblasts from two other kinds of long-lived mutant mice, the Ames dwarf, which also has multiple endocrine abnormalities, and the growth-hormone receptor KO mouse, which has resistance to GH action and therefore low IGF-I levels. These findings show that the stress resistance effect is not limited to a single mutation, single colony, or single background stock. [PubMed]. (2) Resistance of these cells to the DNA damaging agent MMS, and to UV light, suggests that the cells are able to fend off both oxidative and non-oxidative injuries. (3) The difference in stress resistance between cells from Snell dwarf and control cells is not apparent in the first week of life, but develops in the next few months, presumably as a consequence of cellular differentially in an unusual hormonal environment [PubMed]. (4) Cells from mice whose aging rate has been slowed by caloric restriction or methionine deprivation do not show a similar resistance in vitro, suggesting that the mechanisms of lifespan extension induced by these diets may differ from those involved in the dwarf mice [PubMed].
Resistance to oxygen-mediated growth crisis
Campisi and her collaborators have shown that the growth "crisis" typically seen after 5 - 15 doublings in cultured mouse embryonic fibroblasts (MEF), characterized by apoptosis, cessation of net cell production, aneuploidy, and transformation, represents a toxic response to the oxygen levels (20%) used in standard cell cultures. These workers found that culturing MEF at a level of oxygen (~3%) closer to the concentration of post-capillary tissue beds, prevents growth crisis and diminishes the rate of accumulation of mutations and aberrations thought to represent early stages in cellular transformation. We have found [PubMed] that fibroblasts from normal adult skin also exhibit a decline in cell accumulation at passages 5 - 10, which can be delayed considerably by growth in 3% oxygen. Cells from Snell dwarf donors, however, resist these changes in culture growth patterns even when grown at 20% oxygen. This suggests that resistance to oxygen-mediated genetic alterations might contribute to the low age-adjusted tumor incidence in Snell dwarf mice.
Metabolic abnormalities: rotenone and low glucose
In addition to their resistance to death induced by lethal agents, fibroblasts from Snell dwarf mice show resistance to metabolic abnormalities induced by two conditions: exposure to low doses of rotenone, and culture in medium with very low glucose concentrations. Normal fibroblasts are able to reduce extracellular substrates, like the tetrazolium dye WST-1, through a poorly characterized set of plasma membrane proteins and intermediate electron acceptors, known collectively as the Plasma Membrane Redox System or PMRS. Fibroblast reduction of WST-1 is diminished, through unknown pathways, by exposure to rotenone (better known as an inhibitor of the mitochondrial electronic transport chain Complex I), or by culture in medium containing very little glucose. PMRS activity in cells from Snell dwarf donors, however, is resistant both to rotenone and to low glucose media. When cells from normal (non-dwarf) mice are evaluated, those individual cell lines that best resist the effects of low glucose media are most resistant to the lethal effects of peroxide and cadmium, suggesting that the pathways that regulate death after lethal stress and modulate PMRS action in low glucose conditions share overlapping features [PubMed]. Further work on the molecular basis for resistance to low glucose medium and to rotenone may provide new insights into the basis for resistance of Snell dwarf cells to lethal injury as well.
Mechanism of stress resistance in fibroblasts from Snell dwarf mice.
A major current focus is to develop a better understanding of the reasons why fibroblasts from Snell dwarf mice are so resistant to multiple forms of injury in vitro. There are many clues. (1) The fibroblasts from the long-lived mice repair UV-induced DNA damage more rapidly than cells from control mice [PubMed]. (2) These cells also turn on a class of "immediate early genes," including egr-1, fos and jun, more quickly than control cells when exposed to Cd and peroxide stress, but they are relatively slow to activate the stress-activated protein kinease Erk under the same conditions [PubMed]. (3) Cells from the dwarf mice are resistant to membrane lipid oxidation, have relatively high levels of glutathione, consistent with elevated activity of the transcription factor Nrf-2, and indeed many of the mRNAs dependent on Nrf-2 activation are elevated in the Snell dwarf cells [PubMed]. (4) In contrast to their resistance to most forms of lethal insult, cells from the dwarf mice are MORE sensitive than controls to apoptotic death induced by agents that block processing and folding of proteins in the endoplasmic reticulum (the "ER stress" response) [PubMed].
We are also beginning a series of studies to see if the stress resistance of cells from Snell dwarf mice, seen in culture, also affects cells in live mice. An initial experiment, done as a collaboration with Alex Bokov and Arlan Richardson, showed that the liver of Snell dwarf mice differed from control livers in many control pathways relevant to stress resistance (and perhaps to aging), including activation of ERK, JNK, and p38 kinases, expression of genes regulated by Nrf2, and expression of genes that control apoptosis [PubMed]. A study of mRNA for heat shock proteins and related chaperones also found multiple differences in Snell dwarf liver, muscle, heart, and other tissues [PubMed].
Work currently underway focuses on a variety of problems:
- Increased autophagy as a factor protecting Snell dwarf fibroblasts from injury in culture
- Role of ATF4 target proteins in stress resistance
- Modulation of stress resistance by early life exposure to GH and/or IGF-1
Researchers: Scott Leiser, Jim Harper, Liou Sun, Amir Sadighi Akha, Ayesha Rahman, Mike Steinbaugh
Alumni: Scott Leiser, Shin Murakami, Scott Maynard, Amir Sadighi Akha, Adam Salmon.
Support: National Institute on Aging
[last update: December, 2011]
Dwarf mouse (checking the tires) and control (on the vehicle.)
Higher LD50 values for fibroblasts from dwarf mice
Fibroblasts from long-lived rodents are resistant to lethal effects of cadmium
Maximum longevity for various species of rodents