Almost everything we use needs to be repaired at some point in time.We are constantly making the hard decision of whether somethingshould be repaired or replaced. Our genes also need repair. Thedifference is that we can't replace our genes yet, so we have nochoice but to repair them.
When our cells divide, they must make an absolutely accurate copyof our genes. This means that our cellular replication machinery mustfaithfully copy more than 1 billion DNA nucleotides each time a celldivides, a truly daunting task. Also as a result of environmental andother insults to our DNA, cells must repair their existing complementof genes. Otherwise this damage would have far-reaching effects ofgene mutation, tumor formation and cell death.
Mammalian cells have a unique pathway to repair the damageddouble-stranded DNA of our genes. Slowly, but surely, the molecularmechanics that perform these repairs are being uncovered.
In articles published Friday in Science, two groups of researchersdescribe how radiation sensitivity and immune deficiency share thesame defect in expression of an enzyme believed to be involved indouble-stranded DNA repair.
In one report by Susan Lees-Miller and colleagues of the Universityof Calgary and the Cross Cancer Institute in Edmonton titled,"Absence of p350 subunit of DNA-activated protein kinase from aradiosensitive human cell line," they show that a radiation-sensitivecell line did not express the p350 subunit of DNA-dependent proteinkinase (DNA-PK), a potential DNA repair enzyme.
In a companion report by Cordula Kirchgessner and colleagues atStanford University Medical School, Massachusetts General Hospitaland St. John's University in New York titled, "DNA-dependentkinase (p350) as a candidate gene for murine SCID defect," they findthat cells from severe combined immunodeficient (SCID) mice didnot express the p350 subunit of DNA-PK, but that cell hybridscontaining fragments of human chromosome 8 did express thisprotein.
The Common Denominator
Lees-Miller's group used a human malignant glioma cell line that wassensitive to gama irradiation and was effective in DNA double-stranded break repair. When these investigators assayed for DNA-PKin extracts of these cells, they found no activity. When they addedpurified p350 subunit extracts from normal cells, protein kinaseactivity returned. Although these mutant cells did not express thep350 subunit of this protein kinase, the gene's structure appearednormal.
Kirchgessner's group found that the p350 subunit levels in cells fromSCID mice were greatly reduced. These researchers made hybridSCID cells containing various fragments of human chromosomes.They found that p350 was expressed in the cell hybrids containingchromosome 8, implicating this as the site of the p350 gene.
Besides being deficient in the DNA recombination processesnecessary for immune system function, SCID cells are also sensitiveto radiation. By using this latter characteristic, these scientists wereable to show that resistance to radiation was restored in the hybridcells that contained fragments of human chromosome 8 andexpressed p350.
Taken together, these two studies indicate that a defect in theexpression of the p350 subunit of DNA-PK is the basis for bothradiation sensitivity and immune deficiency. As stated in theKirchgessner paper, DNA-PK activates many substrates in vitro byphosphorylation, including transcription factors and proteinsimplicated in the response of cells to DNA damage. This enzymerequires double-stranded DNA for its activity, suggesting that it maybe involved in DNA repair by activating proteins necessary for repairwhile bound to the double-stranded DNA of chromosomes.
Therapeutic Potential For The p350 Gene
Joan Allalunis-Turner, a radiobiologist and senior author of the paperon radiation-sensitive cells, told BioWorld, "This workprogressed so quickly that we are now just catching our breath. Wehave been able to identify a critical pathway in DNA repair and nowwe will be looking at the important upstream and downstreamprocesses in close collaboration with Lees-Miller and RoselineGodbout at the University of Calgary.
"Our immediate goal," stated Allalunis-Turner, "is to look at themolecular mechanism of the defect in p350 subunit expression. Ourwork shows that the gene is present with no apparent rearrangement,but it is not described. Therefore, we believe that the defect is in thepromoter region of the p350 gene."
When BioWorld asked Allalunis-Turner about the therapeuticimplications of her work, she responded, "There is no immediatecommercial application, but it is a mechanism that potentially couldbe used in cancer treatment. Specifically, since many tumors areradiation-insensitive, an understanding of DNA-PK could allow thedesign of an inhibitor that could be useful to treat these tumors."
On the other end of the spectrum, the realization that expression ofthe p350 subunit of DNA-PK can rescue SCID mouse cells from theeffects of this mutation has implications for the treatment of SCID inhumans. Potentially, gene therapy that inserts the p350 gene into theimmune system precursor cells of humans with SCID could reversethe immunodeficiency seen in these individuals. n
-- Chester Bisbee Special To BioWorld
(c) 1997 American Health Consultants. All rights reserved.