In its wild-type native form, p53 performs like a law-and-order white-hat sheriff in the Wild West movies. But p53's mutations _ over 500of them _ convert the tumor-repressing protein into a black-hatscoundrel that turns the tumor loose.

Fully half of all malignancies have mutated p53 lurking in their cells.(See BioWorld Today, Sept. 9, 1994, p. 1.)

Why and how this protective protein defects is aptly explained by apioneer p53 researcher, molecular biologist Richard Iggo, who is atthe Swiss Institute for Experimental Cancer Research, near Lausanne.He was one of the first, perhaps the first, to define p53 as atranscription factor.

"That means," Iggo told BioWorld Today, "that it binds to a piece ofDNA, then activates production or expression of the genes adjacentto it. The gene for p53 itself switches on to stop a redundant oraberrant mammalian cell from growing, or to actively kill it."

Two adjacent genes act as p53's enforcers: "p21," Iggo said, "inhibitsthe several kinases [enzymes] that control the individual steps in thecell cycle. It absolutely locks the cycle, so the cell cannot divide.

"The other target gene, bax by name, sits on the pathway to apoptosis_ cell suicide.

"So when p53 is activated, it either locks the cell cycle and says tothe cell, `You'll never divide again,' or it says, `You're going to die.'"

p53 To Cell: `Okay, You Die!'

"If the cell is going through a division where it makes some terriblemistake _ such as becoming cancerous _ and nothing can be doneabout it, p53 is the final thing that acts before a new cell cyclebegins." Such malignant transformation, Iggo pointed out, "arises notfrom a point mutation, but from a big chromosome-size screw-up,such as chromosomal loss or translocation. When that happens, itactivates p53, which tells that cell, `Okay, you die,' or `Okay, youstop.' "

Once defected from wild type to mutant, Iggo continued, "p53 can nolonger bind DNA. When it can no longer bind DNA, it can no longeractivate transcription, no longer turn on p21 or bax. That's why p53mutations let malignant cells go on dividing _ when they shouldn't."

It's been determined that p53 can be mutated at 542 different nucleicacid sites on its DNA-binding domain. This extends roughly from site100 to 300 on p53's 393 amino-acid length. "The proteins thesemutants encode," he observed, "will inactivate the transcriptionfactor."

About 90 percent of the mutant codons in that binding domain havebeen found in tumors. Iggo cited examples of such cancer-causingp53 aberrations:

* "An exchange of G for T [guanine for thymine] on the DNAsuggests an exogenous carcinogen, such as benzpyrene, that producesadducts [add-on sequences], which are repaired in such a way thatyou end up with G to T.

* "C to T [cytosine to thymine] suggests just spontaneous chemicaldecay of DNA.

* "Skin cancer is caused by sunlight, because ultraviolet radiationproduces mutations where two pyrimidines [uracil, thymine orcytosine] line up side by side in the DNA.

* "In lung cancer, you almost always see G to T [guanine tothymine], given that lung cancer is caused by tobacco, anenvironmental carcinogen. It all makes sense."

Because these mutations can occur at any one of those 542 sites, Iggopointed out, "detecting them is much more difficult than if you wantto look at oncogenes, such as ras for example, which has only two orthree sites."

To overcome this difficulty for clinical purposes, Iggo, who isBritish, with his Swiss and French colleagues, has contrived a simpletest for pinpointing p53 mutations. It's described in the currentProceedings of the National Academy of Sciences (PNAS) datedApril 25 as, "A simple p53 functional assay for screening cell lines,blood and tumors." The paper states that "this form of p53 functionalassay can be used rapidly to detect germline mutations in bloodsamples, somatic mutations in tumors, and mutations to cell lines."

Iggo aproached his assay "from the angle of being a p53 personstudying p53. I pushed it into yeast cells, showed that it worked as atranscription factor, and proposed that you could detect mutations bygap repairing.

"So what you detect," he continued, "is p53 mutants, which cannotactivate transcription."

The trick in enlisting yeast, Iggo said, is "the fantastic efficiency withwhich Saccharomyces cerevisiae performs gap repair in clonedplasmids." Briefly stated, his assay puts PCR-amplified p53 RNAinto a linearized vector, which the yeast cells duly circularize byhomologous recombination. By use of selective markers andenzymatic manipulation, the yeast colonies turn red in the presence ofmutant p53, but stay white to denote the wild-type protein.

Iggo and his former collaborator, Stephen Friend, now at the FredHutchinson Cancer Center in Seattle, are co-inventors of the assay,on which patents are pending. They have licensed the invention toOncor Inc., of Gaithersburg, Md., which in turn has sublicensed it toOncorMed for medical service use. (see BioWorld Today, Oct. 13,1994, p. 2.)

OncorMed's president and chief operating officer, Doug Dolginow,told BioWorld, "We have been using Richard Iggo's colorimetricassay since last summer, and see a steady progression in interest fromphysicians around the country."

He added that "the primary use that individuals have made thus far isin evaluating tumor tissue for the presence of p53 mutations, todetermine prognostic outcomes or chemotherapeutic responses.They've been used in gastric, bladder, colon, breast and braincancers."

Dolginow observed that the assay "replaces straight automatedsequencing for p53, as well as another 10 or so mutation detectionmethods, all of which suffer from various problems."

Charges range from $300 to $400 per test, which is usuallycompleted in under a week.

As for making his assay available for pure research use, Iggo said,"Obviously, people can just write to me and ask _ it's a condition ofsubmitting a paper to PNAS." Oncor, too, is providing it toresearchers.

Iggo concluded, "Probably in the relatively near future people willdevelop cancer treatments that are specific to p53 mutations. Alreadywe know that most standard chemotherapy and radiotherapy workthrough the p53 gene. Obviously for the long term, people are tryingto develop better drugs, specifically for cells where the p53 ismissing.

"And finally gene therapy _ putting back wild-type p53 into the cell.To me, that is where our p53 functional assay will play the biggestpart." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.

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