13 June 1998 AUA98\CADHERIN.MS-- 845 words
PCa resistance gene switches apoptosis off,
altered protein lets immortal factor
fall into nucleus
San Diego--When cancer cells become chemotherapy-resistant, researchers can now find the gene responsible, figure out the mechanism of resistance, and discover another step in the development of cancer. At the 93rd Annual American Urological Association meeting here, researchers from at Columbia University, New York, characterized one more resistance gene in prostate cancer.
This gene creates an altered version of a cadherin protein, explained Ralph Buttyan, MD, associate professor of urology. The cadherin protein normally closes a switch that kills a cancer cell. The altered protein leaves that switch open, and lets the cell proliferate.
"I've always been interested in the genetic basis for hormone resistance in prostate cancer, so we've decided to look at it in a cell culture model," said Dr. Buttyan. That was the LNCaP cell line.
"LNCaP cells can grow in culture, but we can kill them either by treating them with a phorbol ester or by growing them in a medium which lacks serum," said Dr. Buttyan. After four cycles of killing off most of the cells, and selecting the ones that were more resistant to this treatment, they had developed two cell lines. One was LNCaP TR (for TPA resistance), and the other was LNCaP SSR (for serum starvation resistance).
However, each line was resistant not only to the conditions that created it, but to the other conditions as well. The LNCaP line was resistant to serum starvation, and the LNCaP SSR line was resistant to phorbol esters. This indicated a common, basic mechanism of resistance, explained Dr. Buttyan.
The next step was the differential display technique that uses PCR to compare the entire output of messenger RNA in one cell line to the entire output of mRNA in another line. "We did find that there was one genetic difference between the parental cell line and the resistant cell lines," said Dr. Buttyan. "It was a gene product that we called T6." T6 was expressed at very low levels in the parental cell line, and overexpressed in the resistant cell line. It was also found in very high levels in metastatic primary human prostate cancer specimens, but not in non-metastatic specimens.
They sequenced the gene and it turned out to be a new member of the cadherin gene family. "We were very surprised by these results," said Dr. Buttyan, "because cadherin has never before been placed in the apoptotic pathway."
But they found evidence that T6 is participating in apoptosis resistance, said Dr. Buttyan.
Beta-catinin is a protein which helps determine whether a cell will survive, proliferate, or die. Normally, beta-catinin is found in the cell membrane, and the cell is susceptible to apoptosis. But when beta-catinin goes to the nucleus, the cell is resistant to apoptosis--even when it should die, as in tumor proliferation.
Normally, cadherin holds the beta-catinin at the cell membrane, and allows the cell to undergo normal apoptosis, Dr. Buttyan explained. But the T6 cadherin doesn't seem to be able to hold the catinins as well as the normal cadherins. So T6 lets the beta-catinin slip into the nucleus and protect the cell from apoptosis.
The cadherins bind cells to each other. The cells are released under two circumstances, said Dr. Buttyan. First, they're released before the cell undergoes apoptosis. Second, they're released before the cell initiates mitosis. "In order for the cells to divide, they first have to separate from their neighboring cells," he said.
Another urology department, at the University of Michigan, Ann Arbor, similarly discovered two genes associated with metastatic prostate cancer, but they haven't figured out the mechanism yet. The laboratory of professor Kenneth J. Pienta, MD, compared the mRNA from a non-metastatic AT.1 Dunning rat prostate cancer cell line to the mRNA from a metastatic cell line derived from it, the MAT-LyLu (MLL) line.
The MLL line "is a spontaneously metastatic variant of the AT.1 line," said Eric D. Schwab, PhD, assistant research scientist.
They found two complimentary DNA sequences, which they called rPMET-1 and rPMET-2. These two sequences are previously unknown. "They're not found in the GeneBank data base," said Dr. Schwab. However, "we did compare a piece of our gene to the mRNA from genes that are expressed by several standard human prostate cancer cell lines, including LNCaP, and we found a gene of similar size and 700 base pair match" in several lines.
The next step, said Dr. Schwab, is to clone the full-length genes, transfect them back into the AT.1 cell line, and grow them in the rat. "We hope to see an increase in metastases in the AT.1," he said. That will prove that rPMET-1 and rPMET-2 do cause metastases.
"That will give us a better understanding of the metastatic process in general," said Dr. Schwab, "and if we have that understanding, we can use these genes, or the human homologs of these genes, as targets for therapeutic agents, or as diagnostic markers to identify nastier tumors."