By David N. Leff

Remember the case of the bumblebee?

According to the laws of aerodynamics, the bumblebee cannot fly, because the ratio of its wingspread to its body weight makes flight impossible. However, the bumblebee is ignorant of the laws of aerodynamics, and flies anyway.

Now comes the case of a knockout mouse in Ohio that grows up healthy and normal despite lacking a crucial protein that triggers programmed cell death — apoptosis.

In recent years, the key roles played by apoptosis in prenatal development and post-natal disease have become common knowledge. For starters, consider the origin of the immune system.

"Nature has a very elegant way of asking those embryonic immune cells that would recognize 'self' - for example, your heart, your lungs, your own tissues — to die through programmed cell death," observed molecular cell and neurobiologist Ming Xu, at the University of Cincinnati [Ohio]. "Otherwise," he pointed out, "you would really be in trouble."

Xu explained: "In the particular state of immune-system embryonic development, a biological process called negative selection occurs. It eliminates, by apoptosis, those growing immune cells that would recognize self. If this process is incomplete, the individual develops autoimmune diseases. So embryogenesis is one place where apoptosis will play a major role. "Another, he said, would be after an immune response. "For example, if a virus infected your cells, your body would start an immune response to eliminate those abnormal cells. That's where programmed cell death would also be important."

When a cell is thus sentenced to programmed death, the executioner is a protein called the "DNA fragmentation factor" — DFF45. Its name reflects the molecular mechanism by which that death sentence is actually carried out.

"Once a cell is going to die," Xu said, "a series of signal transduction events will be activated inside the cell, aimed at chopping up its DNA into small pieces. To trigger this DNA fragmentation, a protease enzyme, caspase-3, cuts the DFF45 protein, which is then digested. That in turn activates another enzyme, DNA nuclease, which starts cutting the whole chromosomal DNA into small fragments, roughly 200 base pairs long. Finally, neighboring macrophages will engulf and remove this debris."

Xu is senior author of a paper in the current Proceedings of the National Academy of Sciences (PNAS), dated Oct. 13, 1998. It is titled: "Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45."

Astonishingly Counterintuitive Bottom Line

Seeking to pinpoint the precise function of this protein, Xu and his co-authors created a colony of mice lacking the gene for DFF45. What these knockout animals told them was, in his word, "surprising."

"I think the biggest bottom line," Xu told BioWorld Today, "is that when this crucial protein, which is involved in programmed cell death, was destroyed, it did not really affect the general development of the resulting knockout mice. This is quite surprising in two ways. One is, why would nature design this sophisticated genetic program to cut DNA within a dead cell, if without so cutting nothing would happen? This very interesting question really demands that scientists, including our own lab, do more experiments to understand that phenomenon.

"Secondly," he said, "it's really against the conventional thinking. People would think, 'Oh, because nature designed this sophisticated program, there must be something important in there. And therefore by destroying this protein we must find some major consequences.' We did not."

Xu and his co-authors did find, on the contrary, that development of the immune system appeared to be normal in the DFF45-minus mice. Moreover, "they stayed healthy for up to at least eight weeks of age, and none of them developed any apparent abnormalities. We couldn't find any, when compared to the immune cells within a normal wild-type mouse."

Xu and his collaborators are now at work trying to uncover a clue to this apparent paradox.

"When we performed these initial experiments," he explained, "we used relatively young mice. Now we are doing more experiments to see whether older mutant mice develop several kinds of diseases if apoptosis is abolished. Over longer periods of time, these animals would develop tumors or neurological disorders, or the third type, autoimmune diseases. All three of these disease categories, Xu added, "can be summed as the longer-term developmental effects in this particular DFF45 protein. These effects needed to be addressed, and we're in the process of doing that.

Pick Your Paradox-Solving Theory

To account for this unaccountable redundancy of the DFF45 executioner protein, Xu entertains two alternative, tentative hypotheses:

One is that maybe the endogenous system for removing the dead cells actually includes non-digested DNA — because the DNA fragmentation didn't happen. Maybe it's equally effective in removing large and small fragments of DNA.

"Alternatively," he surmised, "this DNA fragmentation is not really necessary. Perhaps it arose by chance during evolution, or maybe there are other functions that we don't know about. It cannot be the case that nature devised this system, which is highly conserved from the round worm all the way to mice and humans, for no good reason." *