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Symposium 21: Beyond Apoptosis: Alternative Responses to Therapy in Tumor Cells

How much is apoptosis involved in the response of solid tumors to therapy?

Stanford University School of Medicine, Stanford, CA

Abstract

SY21-1

The prevalent model to explain how cancer therapies work in recent years has been that anticancer agents trigger the cell to undergo apoptosis, often mediated by p53, and therefore that cells resistant to apoptosis (through such means as overexpression of the anti-apoptotic protein Bcl-2, or mutation of p53) will be resistant to therapy. However, in recent years several investigators have presented data that challenge this paradigm at least for non-hematological malignancies (1–5). In addition no clear pattern has emerged in the clinical literature as to the role of apoptotic genes in the response of cancers to treatment (6). In recent years it has been clearly shown that in addition to apoptosis, cells can be effectively eliminated following DNA damage by necrosis, mitotic catastrophe and by premature senescence, which irreversibly arrests cell division. Of these mitotic catastrophe is probably the most important mode of cell death in tumors following treatment with DNA damaging agents. Whether a cell undergoes mitotic catastrophe and eventually dies (which can be by apoptosis following cell division), depends very much on its ability to repair the DNA lesion which in turn depends on a multitude of gene products involved in the various pathways of DNA repair. However, unlike apoptosis, which is regarded as an active process responding to the trigger produced by the DNA damage, mitotic catastrophe is a stochastic (random) process in a population that is not predicable for any particular cell. We present evidence in support of this with human tumor cells that sustain a translocation in chromosome 4 after 5 Gy of radiation. Only the cells with reciprocal translocations in chromosome 4 are retained while those in which the translocation is non-reciprocal (which produces a dicentric and an acentric fragment), die and are lost from the population. As the probability of a translocation between two broken chromosomes being either a non-lethal reciprocal or a lethal non-reciprocal exchange is 50:50 and almost certainly random, this would argue that death in these cell lines is stochastic after irradiation. The fact that there are multiple forms of death that account for killing cancer cells, and that these individual forms of cell death can occur with different kinetics in different tissues makes assessing cell death complicated. Thus although dead or senescent cells can be readily identified and their proportion in a population measured, it is difficult, especially in vivo, to estimate the total number of cells killed by a specific treatment by examining one time point. Despite this many investigators choose a single time point, such as one or two days after treatment to assess cell death. This is particularly problematic if a particular treatment or genetic manipulation changes the rate at which cells die: by using a fixed time point to assess cell killing, this can be interpreted as changes in levels of cell death even if it is only a change in the kinetics of death. The problem of correctly assessing the fraction of cells surviving a given treatment was solved for mammalian cells in vitro in the mid 1950s by Puck and Markus (7) who developed the technique of cloning individual mammalian cells in vitro . The ability of a single cell to grow into a colony (usually defined as greater than 50 cells) is an assay that tests every cell in the population for its ability to undergo unlimited division. As this assay is carried out a long time after treatment (typically 10 - 20 days for mammalian cells) it integrates all forms of cell death including premature senescence. Though there are caveats to the use of the clonogenic assay, it is the best measure for overall cell death and permanent growth inhibition by DNA damaging agents for most cells in vitro. In addition it can be used to assay the survival of the cells in many experimental tumors by removing the cells after treatment and plating them in vitro, or for a number of normal tissues in which clones of surviving cells can be counted in situ (8). Some, but by no means all, normal tissues are sensitive to the induction of apoptosis by DNA damaging agents. Such cells and tissues include thymocytes, spermatogonia, hair follicle cells, stem cells of the small intestine and bone marrow and tissues in developing embryos. Tumors arising from these tissues, including T cell lymphomas and some hematological tumors, are often sensitive to the induction of apoptosis and experience a marked overall response following treatment with DNA damaging agents. Apoptosis in these tumors is invariably p53-dependent and if inhibited it also changes the sensitivity of the organ or tissue to the DNA damaging agent (4). This is illustrated by the elegant work of Lowe and colleagues with murine lymphomas (9). However, for the majority of tumors, which are of epithelial origin, the primary reason for cell death is not the induction of apoptosis, and although apoptosis can occur in some of these tumors following treatment with DNA damaging agents, it is most likely because of mitotic catastrophe initiated by residual DNA or chromosomal damage. We will show our own data as well as those of others with cells in vitro and tissues in vivo that demonstrate that changing the sensitivity to apoptosis produces little or no change in the overall sensitivity of the cells or tissue to radiation or anticancer drugs. Recent studies of caspase levels in the NCI 60 cell panel and their lack of correlation with the sensitivity of the panel to anticancer agents show that even for 2-day growth inhibition, an endpoint that is highly dependent on the presence of wild-type p53 (10), apoptosis is unlikely to be the reason for the response of this diverse set of cell lines to the anticancer agents (11).