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Researchers Explore Role of Gene Transfer in Tumor Growth

Tracy Webb

While studying angiogenesis in Judah Folkman’s laboratory, Lars Holmgren, Ph.D., and his colleagues noticed that, when angiogenesis was blocked, some tumors maintained their size because tumor cells were dying off as quickly as they were proliferating.

"We knew excessive apoptosis takes place, and we wondered, ‘What happens with all that DNA?’" recalled Holmgren, who is now in the Department of Oncology-Pathology at the Karolinska Institute’s Cancer Center in Stockholm, Sweden.

Holmgren set out to determine if neighboring tumor cells can recycle and reuse tumor DNA from cells that have died off through apoptosis. This recycling, called horizontal gene transfer, occurs when genetic material from a donor cell transfers to and propagates in a recipient cell. Bacteria and fungi use horizontal gene transfer when adapting to new environments and developing drug resistance, and viruses use the same process to propagate in human cell lines.

After a cell dies through apoptosis, the remaining apoptotic body is engulfed by neighboring cells in a housekeeping process called phagocytosis. However, it was generally believed that the DNA was degraded, and therefore inactivated, rendering it harmless to the recipient cell. But is this necessarily the case?

To see if this leftover DNA could be transferred to the recipient cell in a relatively intact form, Holmgren and coworkers demonstrated that HIV-1 DNA isolated from apoptotic cells of HIV-infected patients was taken up by human fetal fibroblast cells that lack the HIV receptor; Epstein-Barr virus DNA could also be taken up by various human cell types that lack receptors for EBV. Both viral DNA and genomic DNA from apoptotic donor cells were taken up and expressed by the recipient cells. In addition, the horizontal transfer of genes was shown to be a surprisingly efficient process.

Genetic Diversity

Two independent laboratories have established that human cells are able to engulf apoptotic bodies in vitro and express the engulfed DNA. But could tumor-promoting genes be transferred through a similar mechanism?

It appears that they can. Holmgren and coworkers found that p53-deficient mouse fibroblast cells can express the oncogenes H-RasV12 and c-myc obtained from apoptotic bodies of rat fibroblast cells transfected with these genes. The recipient cells showed signs of transformation in vitro and formed tumors when implanted into mice. "We found that entire chromosomes were transferred [to the recipient cells], and we also found fusions, or translocations, between tumor cell chromosomes and recipient cell chromosomes," said Holmgren. In addition, the tumor cells isolated from mice expressed the two oncogenes at the protein level.

Holmgren speculated that horizontal gene transfer is indeed one mechanism by which tumor cells generate genetic diversity and thereby promote malignancy. "The initial hypothesis was that the tumor cell accumulates mutations, but we did not know how they accumulated these mutations at such a high rate. These mutations may occur in different cells in the microenvironment and be taken up by phagocytosis," said Holmgren.

In an independent laboratory, Ralph Buttyan, Ph.D., in the Department of Urology at the College of Physicians and Surgeons, Columbia University, New York, and coworkers demonstrated that prostate cancer cells exchange and propagate drug resistance genes in vitro through the engulfment of apoptotic bodies.

Circulating DNA and Genometastasis

It has been known for some time that free, extracellular DNA, originating from apoptotic and necrotic cells, circulates in the plasma and serum of cancer patients and, to a lesser extent, in healthy patients. Some correlative studies suggest that the presence of circulating tumor DNA is associated with metastasis and poor clinical outcome in patients with advanced disease. The potential use of circulating DNA as a diagnostic and prognostic biomarker for cancer, such as EBV DNA in nasopharyngeal carcinoma, is under investigation.

Damian Garcia-Olmo, Ph.D., Autonomous University of Madrid, Spain, and Dolores Garcia-Olmo, Ph.D., Albacete General Hospital, Spain, believe that dominant oncogenes circulating in plasma within apoptotic bodies may promote the development of independent tumors at distant sites as a result of horizontal oncogene transfer, a hypothesis termed "genometastasis."

Although Holmgren believes that horizontal gene transfer may be one mechanism for generating genetic diversity in the primary tumor, he questions the hypothesis of genometastasis. Recent in vitro data generated in his laboratory suggest that normal, healthy cells engulf apoptotic DNA but that this DNA cannot be propagated because of the protective effect of p53. Holmgren believes that apoptotic DNA engulfed by healthy cells may be viewed as damaged, which would trigger p53-mediated growth arrest. In support of this theory, he has found that cells deficient for p21, a downstream mediator of p53-mediated growth arrest, are also vulnerable to transformation induced by engulfed tumor DNA.

Garcia-Olmo said he does not think that p53 protects all cells from the stability of transfection. He said that their experiments have shown that, when plasma from tumor-bearing rats was added to a culture medium, primary fibroblasts were transformed and were able to form tumors in vivo. He added that transfection was not inhibited by the presence of unaltered p53.

Clinical Implications

All of this laboratory research has led to theories about its possible clinical implications. Kathrin Bernt, M.D., a postdoctoral fellow in the laboratory of Andre Lieber, M.D., Ph.D., Department of Medicine at the University of Washington, Seattle, pointed out that chemotherapy can help viruses spread and, conversely, that viral infection can make cells more susceptible to chemotherapy.

Lieber and coworkers, while working on a way to enhance the spread of adenoviral vectors—delivery vehicles for gene therapy—found that inducing apoptosis after viral DNA replication enhanced the spread of the virus among cervical cancer cells in vitro. Through the use of electron microscopy, they demonstrated that neighboring tumor cells engulfed the apoptotic bodies and that these apoptotic bodies contained viral particles. "If you can help the virus escape cells by inducing apoptosis, it can help the spread of the virus because neighboring cells will engulf the virus by phagocytosis of apoptotic bodies," Bernt said.

Chemotherapy may help viral spread by inducing apoptosis. As a result of this work, Bernt said she believes that current adenoviral cancer treatments used in gene therapy may need to be combined with agents that induce apoptosis.

Exploring the possibility of horizontal gene transfer as a means for developing genetic instability and malignancy is still in its infancy. Gene transfer has substantial clinical implications because traditional cancer treatments, namely irradiation and chemotherapy, both induce tumor cell apoptosis, likely enhancing the horizontal gene transfer effect. In support of this, it was recently demonstrated that the antitumor drug mitomycin C induces apoptosis of pulmonary adenocarcinoma cells in vitro with concurrent stimulation of phagocytosis by neighboring tumor cells.

Holmgren believes that these results suggest that chemotherapy may also promote the development of drug resistance. "[Gene transfer] may speed up the phenotype required for resistance during this selection pressure," said Holmgren.

Garcia-Olmo and Holmgren agree that apoptosis-inducing treatments will continue to be used to treat cancer patients. "However," Gargia-Olmo said, "if our hypothesis is verified, a complementary treatment to ‘inactivate’ the circulating nucleic acids should be considered."



             
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