Research At Reed

Mechanisms of Apoptosis

Principal Investigator: Maryanne C. McClellan

Research Assistant: Katherine E. DeLand

Recently, cell death has become a popular topic in the cellular and molecular biology fields and has begun to be recognized as a critical part of both the cellular and whole tissue cycles. Before 1972, all cell death was considered necrosis and there was no attempt made to account for cellular or molecular differences in moribund cells. In a landmark study, pathologists Wyllie, Kerr and Currie defined two types of cell death, necrosis and what they termed 'shrinkage necrosis', which is now known as apoptosis. Necrosis is the consequence of uncontrollable alterations in the cell's environment which cause the immediate compromise of ion transport and therefore, regulation of cell volume. Eventually, due to osmotic pressure, the cell swells and lyses (Wyllie, 1981) which stimulates an immunological response. Apoptosis is a very different sequence of events both morphologically and biochemically.

Apoptosis is programmed, active cell death which requires an alteration of genetic regulation. Although there are undoubtedly multiple biochemical changes in the cell once apoptosis is signaled, the first visible sign of programmed cell death is chromatin condensation in the nucleus (Wyllie, Kerr, and Currie, 1980; Thomas and Bell, 1981). Not necessarily simultaneously, a disturbance in the cytoskeleton causes the cytoplasm to condense around the nucleus which results in nuclear and cytoplasmic material being sequestered in "apoptotic bodies" or blebs without destruction of the organelles (Kinn and Allen, 1981). This occurs without the necrotic release of intracellular material into the interstitial space, again differentiating apoptosis from necrosis. At this stage in the death process many cell types experience cleavage of the genomic DNA into oligonucoleotide fragments of multiples of 180 to 200 base pairs. The final stage of apoptosis is phagocytosis of the apoptotic bodies by neighboring cells or macrophages and break down of the enclosed cellular materials by the lysosomes in the phagocyte which occurs without the immune response seen in necrosis.

At Reed College, Dr. Maryanne McClellan, professor of Biology, specializes in steroid hormone and peptide growth factor regulation of female reproductive tissues. The mammalian reproductive program is characterized by three different hormone controlled cycles, the proliferation, differentiation and regression of the gonads, oviducts, and uterus. In the final stage hormone-dependent regression of both the oviduct and the uterus is associated with apoptosis. If pregnancy does not occur, the additional cells produced in the massive proliferation at the beginning of the cycle are not only energetically expensive but no longer useful and must be deleted. Programmed cell death in response to either steroid hormones or some other endogenous factor is a convenient solution to this problem.

In developing an assay for apoptotic populations in the reproductive tract tissues, we discovered that these cells do not expereience DNA degredation as part of apoptosis. Because early studies indicated that DNA degredation was an essential component of the apoptotic process and because detection of DNA fragments is a relatively simple process, the presence of a DNA 'ladder' of fragments became the hallmark indentifier of apoptosis. Later, it became clear that DNA degredation is not a universal element of apoptosis. Therefore, techniques for identifiying apoptotic populations based on nuclear morphology were developed and are now also considered standard. We decided to persue this avenue of identification.

Figure 1: An image of normal and apoptotic cells captured with a Nikon inverted microscope using NIH Image on a Power Macintosh 6100/60AV.

In this image (which is at 200X magnification), what is shown are the nuclei of feline uteran cells which have been exposed to the steroid hormones estrogen or progesterone and/or the peptide growth factors epidermal growth factor or insulin-like growth factorin vitro. The cells were stained with a DNA binding dye, DAPI, which fluoresces when exposed to ultraviolet light. What is evident from this image is that not all the nuclei exhibit a morphology characteristic of healthy cells. Instead, there are a number of nuclei which are abnormally shaped and some have begun to condense into apoptotic bodies. Once we realized that identification of apoptotis was possible using nuclear morphology, we began to develop a system for quantification. This image was captured using a sensitive camera attached to a microscope with epi-fluorescent equipment. The image was then fed into what is essentially a television and then 'captured' on a Macintosh using a shareware program developed by NIH called NIH Image. Gross counting data were gathered using Image while fine discrimination of healthy versus apoptotic cells was achieved visually.

With the development of this system and the data collected, we have been able to design experiments that allow us to investigate the intracellular signalling of apoptosis in the reproductive tract tissues. We are currently examining lipid based signal transduction pathways and the involvement of cyclic adenosine monophosphate in the genetic alterations required for an apoptotic response.

In a broad sense, these kind of developments are very exciting as they relate to studies of carcinogenesis. Understanding and then gaining control of programmed cell death could be an enormous step in cancer therapy. As Collins and Rivas note, it is likely that tumor therapies will induce tumor cell apoptosis rather than inhibit tumor cell proliferation. Actually, a number of treatments used now, such as radiation and cytotoxin induced DNA damage, can lead to apoptosis (Collins et al., 1992). Unfortunately, these treatments are often less than ideal for the patient as they have numerous side effects and often the targeted tumor cells are not the only affected cellular populations. Eventually, when the signals for hormone dependent cancers, including the aggressive and common breast and ovarian cancers, are better understood, it may be possible to target specifically those cells that proliferate out of turn due to mutation and abrogate, by triggering an alternative genetic program, the progression of tumerogenicity.

Maintained by the Reed College Biology Department
Last Modified 1/09/98
Questions/Comments to yeast@reed.edu