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Does Cancer Therapy Trigger Cell Suicide?

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Science  17 Dec 1999:
Vol. 286, Issue 5448, pp. 2256-2258
DOI: 10.1126/science.286.5448.2256

The notion that drugs and radiation kill cancer cells by causing them to self-destruct has guided drug searches. But some cancer experts aren't convinced

In the dark trenches of the war against cancer, a ray of light seemed to shine through a few years ago: a simple notion about what makes cancers susceptible to radiation or chemotherapy. The key, many cancer researchers came to believe, was the presence or absence of the p53 tumor suppressor gene, which produces a protein that cells need in order to commit suicide when they are damaged or stressed. As long as p53 remained functional, the theory went, cancer cells damaged by radiation or chemotherapy would self-destruct. But if the cascade of genetic changes that led to the cancer also inactivated the p53 gene—which happens in about 50% of all human malignancies—the cells can shrug off the worst that oncologists can throw at them and continue to multiply. As a result, many researchers are looking for ways to restore cancer cells' ability to undergo apoptosis, as cell suicide is more technically called, in order to make them sensitive to cancer therapies.

But some specialists—among them many oncologists who treat tumors with radiation—don't buy this picture, at least not for solid tumors such as cancers of the lung, breast, prostate, and colon. They say that the kinds of test tube assays that point to apoptosis, and p53 in particular, as critical to cancer therapy may not accurately reflect what happens in cancers that have developed naturally in the body. What's more, they argue, using assays based on apoptosis to screen for cancer drugs might cause researchers to miss drugs that kill by other means. “People have become enamored with apoptosis—everything begins and ends with apoptosis—and that's not right,” says radiation oncologist Martin Brown of Stanford University School of Medicine.

Recently, the radiation oncologists have been finding support for their position in a variety of studies showing that p53 gene status does not correlate with a cancer cell's susceptibility to therapy. Not only may cells having a functional p53 gene fail to respond, but those lacking one may be killed easily. But these findings are not easy to interpret, because other genetic factors can also determine whether cells will undergo apoptosis. This makes it difficult to tell whether apoptosis is in fact key to cancer therapy response.

“As with everything in biology and cancer, the situation is much more complicated than we would like,” says cancer gene expert Bert Vogelstein of The Johns Hopkins University School of Medicine. Still, it's critical to learn just what determines whether radiation and drugs will kill cancer cells. As Vogelstein notes, “In the long term, understanding these complexities is likely to enhance our ability to develop better therapies and tailor such therapies to the specific characteristics of individual tumors.”

Apoptosis is a rapid and tidy form of suicide that cells may opt for when their DNA is damaged by radiation or toxic drugs. A great deal of evidence has shown that the decision to self-destruct is controlled by a gene circuit, with the p53 protein serving as the key damage sensor that tells the circuit when to kick in. Over the years several lines of evidence have pointed to the importance of p53-induced apoptosis in determining whether blood cell tumors will respond to treatment.

Pediatric oncologist David Fisher of the Dana-Farber Cancer Institute in Boston cites acute lymphoblastic leukemia, a blood cell cancer that usually afflicts children, as an example. These tumors typically carry an intact p53 gene when they are first diagnosed and virtually melt away when treated with chemotherapy, Fisher says. Recurrences occur in 20% to 40% of cases, however, and when the tumors come back, half of them carry a defective p53 gene and are now resistant to further therapy.

No solid evidence

But although the evidence is convincing for the blood cell tumors, a definitive answer about how p53 influences the response of solid tumors to therapy has been much harder to come by. Simply collecting data from patients doesn't settle the issue, in part because it can be hard to determine whether a cancer has a working p53 protein. Even looking at biopsy samples from primary tumors that are shrinking after treatment to see whether the cells are dying by apoptosis hasn't provided conclusive results because apoptosis is rapid and leaves no traces.

So in 1994 Scott Lowe, then at the Massachusetts Institute of Technology, and colleagues studied experimental tumors in mice. They transplanted cancer cells in which the p53 gene had been inactivated into the animals. The resulting tumors were resistant to both radiation treatments and the chemotherapeutic drug Adriamycin. But tumors formed by cells with an intact p53 gene were sensitive to both types of therapy (Science, 4 November 1994, p. 807). Furthermore, the cells in the shrinking tumors showed such hallmarks of apoptosis as having their DNA chopped into regularly sized bits.

Work on cultured cancer cell lines echoed these findings. Take the National Cancer Institute's (NCI's) drug-screening program, which since 1990 has tested 60,000 potential drugs against a panel of 60 human cancer cell lines. The screening indicated that many current cancer drugs work most effectively in cells with an intact p53 gene, while cells with inactivating p53 mutations tend to resist the compounds (Science, 17 January 1997, p. 343). So entrenched did the idea become that cell biologist Michael Strauss of the Max Delbrück Center for Molecular Medicine in Berlin told the German magazine Der Stern in a 1996 interview that patients whose tumors bear inactivating p53 mutations would not benefit from radiation or drug therapy. Indeed, the interview bore the headline “Jede zweite Therapie ist überflüssig”–that is, “Every second treatment is superfluous.”

Radiation oncologists think that's far too sweeping a conclusion, at least for solid tumors. “We don't worry about doctors,” because they treat patients regardless of their p53 status, says one such clinician, Lester Peters of the Peter McCallum Institute for Cancer Research in Melbourne, Australia. But he adds, “We do worry about patients who might look up the information and decide treatment is futile.”

Stanford's Brown argues that the assays on which Strauss's conclusion was based are flawed. Lowe's transplanted cancer cells were highly artificial, having been made cancerous in the culture dish by the introduction of two oncogenes, E1A and RAS. Many studies, including some by Lowe himself, have shown that these oncogenes raise p53 levels, making cells totter on a knife edge of survival. Thus, Brown says, the cells are poor models for solid tumors because they would never withstand the insults encountered on the way to forming the tumor. Lowe concedes that the model he used was not ideal, but says that finding a model that accurately reflects what happens in natural tumors is a major challenge. “This is the question I've been struggling with for years,” he says.

Brown says that short-term assays like those carried out in the NCI screen to test cancer drugs are also misleading. In those assays, researchers typically bathe cancer cells in high doses of the drug for 2 to 3 days and check the growth response. Cells with intact apoptotic circuitry freeze in their tracks, while those lacking p53 keep going. But the problem is that even if cells survive 48 hours, that doesn't mean they can survive and proliferate in the long term. “p53 [status] tells you how a cell dies but not whether it dies,” Brown maintains.

More than one way to die

He and other radiation oncologists favor a nonapoptotic mechanism, partly because of their clinical experience. The behavior of tumors, they say, seems to indicate that rather than self-destructing, cancer cells die when they try to divide. “Most solid cancers take several weeks to respond [to treatment] because [their cells] have to undergo mitosis,” Peters explains. In fact, he says, one can use a tumor's growth rate to predict how long it will take for a response to become apparent—within weeks for fast growing tumors, whereas slow-growing tumors, such as pituitary adenomas, take years to go away.

More direct evidence for that point of view comes from the so-called clonogenic assays that radiation biologists traditionally use to study the effects of radiation on cancer cells. In these assays, cells are exposed to radiation or drug therapy, seeded onto a culture plate, and then followed for about 12 days, long enough for six or seven divisions. In these assays, cells from solid tumors typically don't die until they divide, at which time the daughter chromosomes break when they try to separate. In other words, death appears to be a consequence of mechanical damage rather than a rapid self-destruct signal.

What's more, these assays don't show a consistent link between cells' ability to undergo apoptosis and their susceptibility to anticancer therapies. In 1995, for example, cell biologist Robert Schimke of Stanford University tested HeLa cells, a line of cultured cells derived from a human ovarian cancer, in both clonogenic and short-term assays. He found that although cells engineered to produce high levels of Bcl-2, a protein that inhibits apoptosis, survived drugs in the short term by resisting apoptosis, they nevertheless had sustained a fatal blow, as evidenced by the fact that they could not form colonies in the clonogenic assay. “Our experiment showed that apoptosis is not necessarily a measure of the success or failure of therapy,” Schimke says. Schimke's experiment has since been followed by a host of others using clonogenic assays, which have come to similar conclusions.

Brown and his colleagues think solid cancers are unlikely even to retain the capacity to undergo apoptosis because of the way they develop. As these tumors grow, they outstrip their blood supply, at least temporarily, leaving their cells deprived of food and oxygen—stresses guaranteed to drive most cells to press the self-destruct button. So only those cells that have disabled their apoptosis circuitry are likely to make it to the point of forming solid tumors. If so, radiation and drugs that shrink solid tumors must therefore be acting by other mechanisms.

Yet the issue is far from settled. Many biologists say the clonogenic assays are as unnatural as the short-term tests that pick up apoptosis. Cells individually seeded onto a plate to test their growth are a far cry from cells growing in a tissue, where their fate may be influenced by contact with neighboring cells or the extracellular matrix. For instance, Caroline Dive and John Hickman of the University of Manchester in the United Kingdom found that lymphoma cells made resistant to apoptosis by an extra copy of the bcl-2 gene had no long-term survival advantage in a standard clonogenic assay. But when the culture dishes were made to resemble tissue by coating them with the extracellular matrix protein laminin and adding the growth factor IL-4, apoptosis-resistant cells had a survival edge.

Further complicating efforts to untangle the situation is the fact that cells' responses to therapy may vary depending on the drug used. One recent example comes from the Vogelstein team at Johns Hopkins. In experiments reported in the August issue of the Journal of Clinical Investigation, the researchers specifically inactivated the p53 gene in a line of cultured colon cancer cells. Although the mutants did become resistant to 5-fluorouracil, a drug widely used to treat colon cancer, they became more sensitive to another cancer drug, Adriamycin, and to gamma radiation.

In addition, p53 status by itself is not enough to indicate whether cells are capable of apoptosis. Other components of the apoptosis circuit can determine the final outcome. For example, mutations that activate the oncogenic potential of Bcl-2 and its relatives are well-known derailers of apoptosis. And recent work shows that the second most common mutation in solid cancers—disruption of the chromosome locus that includes the p19 tumor suppressor gene—also results in the failure of apoptosis.

In work reported in the 15 October issue of Genes and Development, Lowe, now at Cold Spring Harbor Laboratory on Long Island, Clemens Schmitt (also of Cold Spring Harbor), and their colleagues inactivated the p19 gene in a strain of mice already prone to B cell lymphomas because the animals carry an active myc oncogene. The researchers found that the resulting animals developed an aggressive lymphoma that closely resembles the cancers seen in animals with inactivating p53 mutations; among other things, they were highly resistant to chemotherapy. These results mean that researchers wanting to establish whether apoptosis is important in how cancer cells die will have to determine exactly which genes are defective in resistant cells, an effort already going on under the aegis of the NCI in Bethesda, Maryland.

“Brown is right in saying the answer's not known yet; we have to bite the bullet and get into these experiments,” says Dive. The hope is that order will soon emerge from the chaos, says Vogelstein: “Whether the models we have now are correct is not as important as the fact that cancer researchers are for the first time getting some real insights into why drugs fail, and more importantly, why they work at all.”

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