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Columbia University Health Sciences researchers have found a new enzyme involved in arresting the growth of human malignant melanoma and other cancer cells.

The enzyme, called human polynucleotide phosphorylase or hPNPase-old-35, helps induce cancerous cells to lose growth potential and revert to a benign state in their development, a process called terminal cell differentiation. The enzyme also is important in cellular senescence, when a cell cannot divide anymore and dies.

The researchers, led by Dr. Paul B. Fisher, professor of clinical pathology at P&S and director of neuro-oncology research, found the enzyme because they suspected that some of the same genes are involved in cell aging and in how a cancer cell terminally differentiates. Both processes have many overlapping traits, such as growth arrest and changes in gene expression profiles.

"We wanted to know what pathways might be similar between cells terminally differentiated vs. cells that are senescent," says Dr. Fisher, who is also the Michael and Stella Chernow Urological Cancer Research Scientist. The research was reported in the Dec. 23 Proceedings of the National Academy of Sciences.

Other authors on the paper are Dr. Magdalena Leszczyniecka, lead author and formerly a graduate student at New York University; three members of Dr. Fisher's lab: Dr. Dong-chul Kang, associate research scientist at P&S; Dr. Devanand Sarkar, postdoctoral research scientist at P&S; and Dr. Zao-zhong Su, research scientist at P&S; Dr. Kristoffer Valerie, professor of radiation oncology at Virginia Commonwealth University; and Matthew Holmes, Dr. Valerie's graduate student.

To find which genes overlapped, the researchers looked at the mRNA molecules produced by both terminally differentiated and senescent cells. By identifying which mRNAs were common to both types of cells, the researchers could then figure out which genes were involved by tracing the mRNAs back to the genes. This gene identification strategy—the overlapping pathway screening (OPS) approach—also can be used to identify common gene expression changes that occur during other physiological changes in cells, such as growth suppression and response to chemotherapy, chemotherapy vs. radiation, and reversible vs. terminal differentiation.

To get the mRNA profile of the terminally differentiated cells, the researchers stopped the growth of human melanoma cells. Dr. Fisher and his colleagues treated the metastatic melanoma cells with fibroblast interferon and mezerin (a protein kinase C activator) to induce the cells to stop growing.

After removing the entire set of mRNAs from growth-arrested cancer cells and from actively cancerous cells, the researchers subtracted the mRNAs common to both types of cells. The subtraction process left the mRNAs important in terminal differentiation, which Dr. Fisher's team turned into cDNA.

To obtain senescent cell mRNA, Dr. Fisher used skin fibroblast cells from people with progeria because normal cells under these conditions age slowly in culture. Progeria is a premature aging syndrome where, for example, cells of an 8-year-old child with the disease can resemble those of a 70-year-old adult.

The researchers used the progeria cell mRNA as a probe to identify the corresponding cDNAs in the library made from the mRNAs of terminally differentiated cells. The comparison showed an overlap of 75 genes. The investigators chose one of the genes, which they named old-35, for further evaluation because it showed an elevated expression pattern associated with both induction of cancer cell terminal differentiation and the progeria-type senescence.

After checking gene registry databases, they found that old-35 had not been identified and cloned before in humans, although a similar enzyme, PNPase, had been identified in bacteria. hPNPase-old-35 codes for an enzyme that degrades RNA. Dr. Fisher plans to publish another paper about the relationship of the human and the bacterial versions of the gene. He and his lab have filed for a patent on the human gene and its function.

It is unclear what role hPNPase-old-35 plays in a normal cell. It does not appear to be part of an apoptotic pathway, Dr. Fisher says. But the hPNPase-old-35 enzyme could prove to be a powerful weapon against cancer. By selectively expressing the gene in tumor cells, mRNAs that promote growth and survival could be targeted for degradation. By destroying critical mRNAs, cancer cells could be induced to undergo programmed cell death (apoptosis), Dr. Fisher speculates.

The research was supported by grants from the National Institutes of Health, the Samuel Waxman Cancer Research Foundation, and the Chernow Endowment.


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