NEWS

As Easy as ABC: Scientists Fish Out Another Drug Resistance Gene

Bob Kuska

For years, most scientists thought they had an open-and-shut-case to explain why human tumors often grow resistant to chemotherapy. They had assembled a formidable body of laboratory and clinical evidence that all seemed to point to the transporter P-glycoprotein, or PGP, as the sole culprit in triggering chemoresistance. Discover how PGP pumps compounds out of cells to make them resistant to chemotherapy, discover a possible cure for cancer.

Or so the thinking went. But in 1992, the case for PGP hit a snag when Susan Cole, Ph.D., and her colleagues at Queen's University in Canada, pulled out a second transporter called MRP, a cousin to PGP, that the group showed also pumped out certain chemotherapy drugs from an established chemoresistant tumor cell line. As often happens in biology, a seemingly simple story suddenly had turned complex.

Now, three teams of scientists independent of each other report that they have identified another cousin to PGP involved in chemoresistance. The groups — one from the Greenebaum Cancer Center of the University of Maryland in Baltimore and two from the National Cancer Institute — said they found the transporter in chemoresistant cell lines with low levels of PGP and MRP, but with high levels of drug transport, particularly for mitoxantrone, allowing the cells to escape the deadly effects of chemotherapy.

The scientists noted that their in vitro findings must be confirmed in human tumor specimens to confirm the role of this new protein in chemoresistance. In the meantime, they say that, like the MRP finding 7 years ago, this new discovery hammers home the point that better days are ahead in grasping the complex biology of drug resistance.

"I think it is a natural tendency that people try to simplify things, as happened with P-glycoprotein," said Douglas Ross, M.D., Ph.D., of the Greenebaum Cancer Center and author on the recent paper that reported the new transporter. "But now that we are actually starting to unravel chemoresistance in its complexity, I think it will allow us eventually to gain a fundamental understanding of the process."

ABC Genes

Like so many joint discoveries, the new transporter wears numerous acronyms that can make any discussion of it confusing. The group from the Greenebaum Cancer Center, led by Ross and Austin Doyle, M.D., named the protein BCRP; the NCI group, led by Susan Bates, M.D. and Antonio Tito Fojo, M.D., Ph.D., chose MXR; and Michael Dean, Ph.D., also with NCI, opted for ABCP.



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Dr. Austin Doyle

 


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Dr. Susan Bates

 


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Dr. Michael Dean

 
Whatever the final designation may be in the scientific literature, both groups say BCRP/MXR/ABCP is the latest addition to a much studied family of genes, called ABC transporter genes. "ABC" stands for ATP-Binding Cassette, a reference to genes that contain a similar signature DNA coding sequence that allows its protein product to bind ATP in the cytoplasm as its energy source, sort of like an old-fashioned mill along a river taps the constant flow of water for power.

Although ABC proteins perform a dizzying array of tasks in organisms as diverse as humans and bacteria, members of some ABC subfamilies have evolved into cellular freight systems, employing their unique ATP-binding ability at the cell membrane to haul a variety of essential molecules in or waste products out. Given the importance of these tasks to life, ABC genes have been implicated in various human genetic diseases including cystic fibrosis and an eye disease of the retina.

ABC genes also are implicated in chemoresistance. This first came to light in 1976 with the finding that PGP was overproduced in a tumor cell line resistant to chemotherapy. While scientists at first envisioned the simple one-step solution of stifling PGP to improve chemotherapy, the subsequent discoveries of additional ABC genes — MRP, MOAT, Tap 1, Tap 2 — as possible players in chemoresistance, led several laboratories to hunt for more of the estimated 75 or so ABC genes stitched into the human genome.

EST Skimming

One was Dean's laboratory at the NCI in Frederick, Md. In 1996, a postdoctoral fellow in Dean's group, Rando Allikmets, Ph.D., searched a computerized database of known ESTs, or partial sequences of genes expressed in various tissues, looking for sequences similar to a signature ATP-binding motif found in the known ABC gene. The idea being, if there is a match, it might be an ABC gene.

Allikmets, Dean, and colleagues hit the jackpot. As published that year in Human Molecular Genetics, the group reported the discovery of 21 new ABC genes, upping in one article the number of known human ABC genes to about 30. Today, at least 50 human ABC genes have been found.

Included in their take was a partial sequence for a gene with an unusual expression pattern in the body. "When we did expression studies, we found that it was very, very highly expressed in the placenta and nowhere else," said Dean. "We figured it had to be important, but we really didn't have much more than that as to what it was doing." With so many genes to study, however, Dean said his group reluctantly decided to put what they would later name ABCP, for ABC placenta, on the backburner.

Unbeknowst to Dean or anyone else at the time, they had discovered and briefly pushed aside the very gene that his long-time NCI collaborators, Bates and Fojo, had been unsuccessfully searching for in vitro. As Bates recalled, the search for the gene started in the early 1990s after she and Fojo selected a breast cancer cell line with a unique chemoresistance profile, meaning it was resistant to some drugs and not others, but it did not overexpress MRP or PGP. The implication being, there was an unknown transporter in there somewhere. They just had to find it.

"When the cell line was selected 10 years ago, every other clone that was picked got contaminated with yeast except this one," said Bates of the cell line, noted for its resistance to mitoxantrone and Adriamycin. "Lyn Mickley, a biologist in the lab, said, `This is a chosen cell line.' So, we started calling it `The Chosen' in the lab."

But the cell line proved not to be so choice. The two groups had spent a lot of time chasing down false leads, and Fojo said the groups reached a tough decision when a tumor cell line called S1M1, created by Lee Greenberger, Ph.D., at Wyeth Ayerst Research in Pearl River, N.Y., reached their labs showing a phenotype nearly identical to that of The Chosen. Fojo said he and Bates finally decided to go with S1M1, thinking it might be easier for them to fish out the mystery transporter gene.

Meanwhile, Fojo and Bates already had sent their cell line to Ross at the Greenebaum Cancer Center. As Ross recalled, he had been intrigued by earlier work of the NCI group reporting the tumor cell line's mitoxantrone-resistance phenotype and an uncharacterized protein as the possible transporter. "Austin Doyle and I were very interested in this cell line," said Ross, noting that his research focused on multidrug resistance in leukemia patients and mitoxantrone had recently been approved as a treatment for certain leukemias.

Ross said the candidate protein turned out to be a false lead. But he said using differential display techniques to measure levels of gene expression, he and his colleagues eventually fished out a gene sequence that was overexpressed in The Chosen. This led to pulling out and sequencing a full-length cDNA that told Ross and his colleagues that they were looking for a small transporter. "When we saw the size, we were concerned that this was a half transporter," said Ross, indicating that a half transporter is not considered functional unless it teams with a partner protein to make it a full transporter. They went ahead anyway and transfected a full-length cDNA of this transporter into the original drug-sensitive cell line to see whether it could confer drug resistance. Ross added, "Because the transporter was isolated from a multidrug resistant breast cancer cell, we called it at least tentatively, breast cancer resistance protein, or BCRP."

When Ross and colleagues finished their transfection studies, he said the group had enough data to show conclusively that forced expression of BCRP caused the MCF7 cell line to be drug resistant. His group presented a poster of their work last spring at the annual meeting of the American Association for Cancer Research and last December published their findings.

Meanwhile, last summer, Bates and Fojo were closing in on ABCP. Screening a library of cloned cDNA segments isolated from the S1M1 cell line, which can give an indication of the level that a gene is expressed by a cell, they eventually pulled out three overexpressed clones. "Two of the three were for a gene we named mitoxantrone resistance protein or MXR," said Bates.

"When we got this sequence and put it into Genbank, we saw that it was one of Mike Dean's, who we had been collaborating with all along, and we called him," said Fojo. "Our thinking was there was an ABC transporter to be found."

Dean quickly pulled ABCP off the backburner. In December 1998, he and his colleagues published in Cancer Research characterizing MXR/ABCP as being expressed primarily in the placenta. A few weeks later, Dean, Bates, Fojo and coworkers also published in Cancer Research their finding that MXR/ABCP is overexpressed in mitoxantrone-resistant cells.

Lessons Learned

According to Bates, one of the most interesting aspects of the new transporter is that it introduces half transporters as possible players in causing drug resistance. She said this is an interesting issue because half transporters can dimerize, or connect, with other half transporters, possibly creating new combinations of full transporters. The idea being, almost like combining different Lego blocks, the possible number of variables grows. "The diversity [of transporters] just magnifies enormously," said Bates. "The variables grow, and how a cancer cell could use this to its benefit just becomes even more complex."

Another key question is why a tumor cell would switch on a gene that is expressed at low levels in adult tissues, but at high levels in the placenta? Dean speculated that the answer might be as simple as because the gene is there. "I think cells naturally have really sophisticated mechanisms to deal with all of the different toxic compounds that they encounter, and what happens is tumors are taking advantage of these properties," said Dean, who noted that PGP and MRP are also expressed at significant levels in the placenta. "Basically, the genes are there to handle probably any toxic compound that we can throw at tumors. It is just a matter of the tumor turning on the right gene either to inactivate the drug or stop responding to the DNA damage and pump it out of the cell."

Ross said he agrees that BCRP/MXR/ABCP offers some perspective on the complexity of drug resistance. "What this is pointing toward is some idea of complexity and repertory of resources available to a cell to protect itself when stressed," he said. "This could be good in terms of deciphering how to get around those mechanisms in dealing with cancer."

Ross added the reverse might be true, too. He said because the expression level of BCRP/MXR/ABCP is so low in adult tissues, it might be an excellent candidate to target an inhibitor and enhance the effect of the chemotherapy without affecting normal cells. According to several scientists, candidate inhibitors already have been identified.

But, the more immediate challenge is to learn more about the structure and function of BCRP/MXR/ABCP. On this point, Fojo said he sees many positives. "If you look at the life cycle of MRP, the data that were accumulated to bring it up to speed with PGP was about one-third of the [research] time as for PGP. For this one, I think it will be cut down even further. Number one, you learn from past history — how to do it, where to look, what's important, and so forth. But number two, there is just a lot more modern technology available to speed things along."



             
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