Ming Chow

Random population-wide genetic damage induced in replicating cells treated with methotrexate.

Low lethality treatment of the NIH 3T3 mouse cell line with methotrexate (MTX) during exponential multiplication results in heterogeneous, heritable reduction in growth rate of most if not all the replicatively surviving cells. The effective concentrations of MTX are 10 to 100 times higher in molecular, cellular and developmental biology medium 402 (MCDB 402) than in Dulbeccos modification of Eagles medium (DMEM) medium because of the folate-sparing presence of adenine, thymidine and, particularly, of folinic acid in MCDB 402 medium. The reduced growth rates are detectable during early passages of surviving populations before the faster growing cells dominate them. The heritable effect is most clearly demonstrated by sequestered cloning of many individual cells immediately after drug treatment, and repeatedly measuring the growth rates of the clones in serial passages. After 7-10 passages of the clones, there is an increase in growth rate of some of the slow growing clones presumably due to the generation and selection of faster growing cells. Evidence from mutagenic studies at a single genetic locus in other cell lines suggests that heritable reductions in growth rate arise from chromosome aberrations although point mutations may also contribute to the effect. Clastogenic changes can be induced by a wide variety of mutagens and carcinogens, many of which are used in chemotherapy of cancer and other chronic diseases. The population-wide, heritable damage to cells may be the source of, or may contribute to, late-occurring side effects of treatment in cancer and other chronic diseases.

Evidence for cellular aging in long term confluent cultures: heritable impairment of proliferation, accumulation of age pigments and their loss in neoplastic transformation

Previous experiments had shown that repeated rounds of prolonged growth constraint at confluence of NIH 3T3 sublines result in persistent changes in the growth behavior that are characteristic of cellular aging. These changes, which include an enduring decrease in the rate of proliferation in low density subcultures and a marked increase in neoplastic transformation, are here reproduced cumulatively over a 6 week period during which cultures are maintained in a single, continuous round of constraint at confluence. By testing multiple cultures at weekly intervals we show that the persistent reduction in exponential growth in low density subcultures is a property of the entire treated cell population that is first demonstrable in the cell population used here within a few days after the constraint of confluence is imposed. There is also a reduction in saturation density of cells subcultured from this early confluence which is reversed in longer term confluence when the cells become transformed. The reduction in exponential growth rate in serial subcultures becomes more pronounced in cells after longer periods of confluence. It is strongly manifest at 6 weeks when most of the cells have undergone neoplastic transformation. The transformation initially involves only a very small fraction of cells in a confluent culture, and is only detectable after 3 weeks of confluence. Beyond that time there is selective overgrowth of the transformed cells so they become the dominant element at 6 weeks. The very same cells from the 6 week cultures that have a reduced rate of growth when subcultured at low density, grow to higher saturation densities at confluence. The reduced growth rates are heterogeneously distributed among clones derived from the 6 week confluent cultures. Typical age pigment bodies appear in the cytoplasm of the cells after 3-4 days of confluence, and fill the cytoplasm at 2 weeks. They tend to enlarge into residual bodies at 3 weeks but largely disappear at 6 weeks when most of the cells are transformed. The results reinforce the conclusion that the prolonged constraint of confluence of these cells reproduces the major growth and morphological effects of cellular aging in the body.

Relation of the slow growth phenotype to neoplastic transformation: possible significance for human cancer.

SummaryDeletions are widely distributed over the genome in the most frequently occurring human cancers and are the most abundant genetic lesion found there. Deletions are highly correlated with the slow growth phenotype of mutated animal and human cells and result in chromosomal transposition when the retained ends are joined. Transpositions are only a minor source of mutation in rapidly multiplying bacteria but are a major cause of mutations in stationary bacteria. The NIH 3T3 line of mouse cells undergoes neoplastic transformation during prolonged incubation in a stationary state and expresses the slow growth phenotype on serial subculture at low density, suggesting a relation between transformation and chromosomal deletions. To further explore the relation between neoplastic transformation and the slow growth phenotype as a surrogate for deletions, two sublines of the NIH 3T3 cells with differing competence for transformation were serially subcultured in the stationary state at confluence and tested at each subculture for transformation and growth rate. Cell death in a fraction of the population and a heritable slowdown in proliferation of most of the survivors became increasingly pronounced with successive rounds of confluence. The reduction in growth rate was not proportional to the degree of transformation of the cultures, but all of the transformed cultures were slow growers at low density. All of the discrete colonies from cloning transformed cultures developed at a lower initial rate than control colonies under optimal conditions for growth, but they continued to grow at later stages, forming multilayered colonies under conditions that inhibited the further growth of the control colonies. The results suggest that prolonged incubation of NIH 3T3 cells in the stationary state results in growth-impairing deletions over a wide range of sites in the genome, but more restricted subsets of such lesions are responsible for neoplastic transformation. These findings provide dynamic, functional support in culture for the histopathological evidence that the quiescent state of cells associated with atrophy and fibrosis plays a significant role in the origin of some cancers in experimental animals and human beings.

Umasking large and persistent reductions in proliferation rate of aging cells

SummaryWe have reported that nontransformed sublines of NIH 3T3 cells that are incubated under the growth constraint of confluence for 10 d or longer exhibit heritable reductions of growth rate upon serial subculture at low density, which simulate the effects of aging in vivo on cell growth. There is also a marked increase in the likelihood of neoplastic transformation. After switching to a new batch of calf serum (CS), we found the reduced growth rate was no longer produced within the previously established timeframe. However, substitution of fetal bovine serum (FBS) for CS during the period of recovery from confluence or the following tests of growth rate resulted in profound inhibition of growth in cells serially subcultured from confluent cultures. In some cases, fewer than one in a thousand cells from subcultures of confluent cultures formed colonies in FBS although they cloned at relatively high efficiency in CS. The reduced growth in FBS was retained in the postconfluent subcultures after many generations of multiplication at low density in CS. Generally, similar results with individual variations were obtained with three other batches of FBS. The numbers of cells per 3-d colony initiated from subcultures of confluent cultures were lower than those of control cultures that had never been confluent. Supplementation of FBS-containing medium with CS fully restored the growth of the postconfluent subcultures to the rate in CS medium, indicating that there is a deficiency of growth factor(s) in FBS rather than the presence of an inhibitor. The results show that prolonged incubation at confluence induces a populationwide heritable increase in requirement for growth factor(s) in short supply in FBS. Because clonal studies have shown that the reduction in growth rate is irreversible and varies in degree from clone to clone, we propose it arises from damage to DNA at any of many different genetic loci or from chromosome aberrations. Such genetic damage is also consistent with the increased tendency for neoplastic transformation in subcultures from the long-term confluent cultures.

QUANTITATIVE ASPECTS OF THE SELECTIVE KILLING OF TRANSFORMED CELLS BY METHOTREXATE IN THE PRESENCE OF LEUCOVORIN

SummaryA quantitative study was made of the cytotoxicity of methotrexate (MTX) for nontransformed and transformed NIH 3T3 cells in the presence and absence of leucovorin. The study was preceded by an analysis of the growth rates of the cells at low and high population density combined with low and high concentrations of calf serum (CS). The reduced maximal growth rates of the transformed cells at low population densities relative to the nontransformed cells reinforced earlier evidence that heritable damage involving chromosome aberrations drives the process of transformation. When small numbers of transformed cells are cocultured with a large excess of nontransformed cells in the assay for transformed foci, the transformed cells were more readily killed by MTX than the nontransformed cells. The selectivity was increased when leucovorin (folinic acid) was present in the medium. The selective killing of the transformed cells actively multiplying in foci was most pronounced when the background of nontransformed cells had become confluent and their growth was inhibited. However, selectivity has also been demonstrated when transformed and nontransformed cells are growing at their maximum rates at low density despite the lower growth rate of the transformed cells under these conditions. The sensitivity of transformed cells in pure culture to MTX was lower during the first 3 d of subculture than in the following 6 d but decreased to zero a few d after net growth had ceased. The nontransformed cells were more susceptible to killing by MTX in Dulbecco’s modified Eagle’s medium (DMEM) than in MCDB 402, but the transformed cells were sensitive to MTX in both media. The high selectivity of MTX for transformed over nontransformed cells in MCDB 402 results from the presence of 1.0 µM leucovorin (5-formyltetrahydrofolate), a reduced form of the folic acid present in most other culture media. When leucovorin was added to DMEM with its high concentration of folic acid, the resistance to MTX of both nontransformed and transformed cells was greatly increased, but the selectivity of MTX for transformed cells was almost entirely lost. The results indicate that leucovorin protects nontransformed cells against concentrations of MTX that kill transformed cells, but the protection is dependent on the relative amounts of leucovorin to folic acid in the medium. The relative sensitivities of transformed and nontransformed cells in our system to MTX when both cell types are exhibiting their characteristic differential in growth behavior is similar to that described for tumor and normal cells in vivo. Since the unregulated growth behavior of the transformed, tumor-producing cells is efficiently and quantitatively measured in this system, it can be used to develop general principles of treatment and resolve questions of cytotoxic mechanism.