In the February 2006 issue of Cancer Cell, researchers from Columbia University in New York report on the results of a screening study aimed to identify compounds that would help overcome cancer cells’ resistance to p53-mediated cell death - specifically, to the reduction in p53 levels caused by the viral oncogene E6.

The researchers found no fewer than five classes of compounds that were able to restore sensitivity to the anticancer drug doxorubicin (marketed as Doxil by Ortho Biotech, of Bridgewater, N.J., a unit of Johnson & Johnson); what they did not find was any compounds that acted on p53.

"We thought we would find compounds that restore p53 levels. But in fact, none of the compounds we found had that effect," senior author Brent Stockwell told BioWorld Today.

He added that "such serendipity is one of the advantages of the screening approach. We allow the experiment to tell us the interesting mechanisms, rather than trying to guess them ahead of time."

Under normal conditions, the p53 protein is unstable and found only at very low levels in the cell. But when DNA damage occurs, p53 levels rise and either initiate protective measures or apoptosis, depending on the severity of the damage.

P53 is the target of a number of drug development efforts by biotechnology companies including Cleveland Clinic spin-off Cleveland BioLabs; Austin, Texas-based Introgen Therapeutics Inc.; Gemin X Biotechnologies Inc., of Montreal; and Reata Discovery Inc., of Dallas.

P53 function is decreased in more than half of all human cancers. The cause most often is a straightforward mutation in the gene itself, but in some cases, other proteins reduce p53 levels in the cell. The E6 protein, which comes courtesy of the cervical cancer-causing human papilloma virus, is one such protein; it works by tagging p53 for destruction by the proteasome.

In the Cancer Cell study, the researchers used a colon cancer cell line expressing E6 protein to find drugs that would potentiate the effects of doxorubicin. Asked for the reasoning behind using a colon cancer cell line to study a cervical cancer protein, Stockwell explained that "we wanted an isogenic cell system, that is, two cell lines that are genetically identical except for the change that we induce. Also, the parental cell lines need to express p53 in abundance." There were no cervical cancer cell lines available that met these requirements, though the scientists did test all the compounds they identified during screening on cervical cancer cell lines and found they were effective in those cell lines as well.

Doxorubicin binds to topoisomerase II and promotes cell death via several molecular mechanisms, one of which is the stabilization of p53. But as cancer cells acquire mutations that lead to the loss of p53, doxorubicin becomes less effective. Among the five compound types that were effective in restoring doxorubicin’s effects were protein synthesis inhibitors and a class of compounds the scientists named indoxins (because they increase doxorubicin’s effects).

Stockwell and his colleagues were most interested in the indoxins because they selectively increased the effectiveness of doxorubicin in several cell lines. A series of experiments showed that indoxins did not restore cellular p53 levels. Instead, they appeared to work via two separate mechanisms: They upregulated topoisomerase II and induced cell cycle arrest in S phase - the cell cycle phase where doxorubicin is most lethal.

A first attempt to find the molecular target of indoxins using biotin labeling struck out; the binding of indoxins to their targets proved too weak to use this approach. Undaunted, the researchers next tried photolabeling, or adding a tag that can be activated with light and will trap any compound it is interacting with. Using a photolabel in combination with a fluorescein tag, they found that two actin-related proteins, myosin 1c and ARP2, are the indoxins’ main targets.

Though the two proteins are part of a single molecular complex, Stockwell said there is a reasonable explanation for how they might cause both topoisomerase II upregulation and S-phase arrest. "Our speculation - and it is only speculation at this point - is that indoxins target the nuclear myosin 1c that upregulates topoisomerase, but also cytoplasmic myosins that induce cell cycle arrest," Stockwell said.

Asked whether he intends to see this work through into the clinic, Stockwell said that "our work is, in this case, and has always been, about basic mechanisms. But we are always happy to work with industrial collaborators who are interested in pursuing clinical development."

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