Science Editor

Structurally speaking, telomeres are the ends of chromosomes, and functionally speaking, telomere damage is supposed to be the end of cells - it is one signal that causes normal cells to stop dividing and enter into senescence or apoptosis. In other words, death.

Malignant cells somehow manage to circumvent that and other cellular safety mechanisms, allowing them to proliferate long past their expiration date, to the detriment of the now-cancerous organism.

In research published in the September 2004 issue of the Federation of American Societies for Experimental Biology Journal (FASEB), Neelu Puri and colleagues at the Boston University School of Medicine report that the artificial analogues of telomere overhangs can induce apoptosis in malignant melanoma cell lines and reduce primary tumors, as well as metastases in mice.

Tuck That In, You Look Like A Slob

Telomeres do not code for genes; they consist of tandem repeats of the sequence TTAGGG. On the 3-prime end of the strand, there is an overhang of nucleotides. Barbara Gilchrest, professor and chairman of dermatology at BU's School of Medicine and senior author of the study, calls the telomere "a sponge for DNA damage": guanine residues, which make up half of its sequence, are the principal target for oxidative damage, whereas adjacent thymidines, which make up another third of the structure, are the sequence that is most easily damaged by ultraviolet rays.

Telomere structure apparently is critical for its sentinel role. The 3-prime overhang of the telomere is usually neatly tucked back in on the chromosome in a loop structure. DNA damage causes the chromosome to become so slovenly, the overhang flaps about. That, in turn, signals to the cell that it might either be time to repair its genetic code, or fold the cards altogether.

"Exposure of the single-stranded overhang is used by the cell to recognize that DNA has been damaged," Gilchrest told BioWorld Today. "So, if you created an oligonucleotide that had that overhang and you gave it to a cell, would that cell act as if its DNA had been damaged?"

To test that question, the researchers used an oligonucleotide homologous to the 3-prime overhang (the T-oligo), as well as control oligonucleotides with different sequences. They first treated cultures of several melanoma cell lines, as well as control melanocytes, with the T-oligos. They found that T-oligos accumulated in the cell nucleus and caused melanoma cells to undergo apoptosis. Control melanocytes treated with the same oligos underwent a brief period of growth arrest, but soon resumed normal activities.

Gilchrest's group then examined the cells histologically to study changes in protein expression in the T-oligo-treated cells. Several proteins that are being clinically investigated as melanoma vaccine targets were up-regulated, including two proteins: MART-1 and gp100. In contrast, the protein livin, which is anti-apoptotic and known to play a role in mediating melanoma cell resistance to chemotherapy, was down-regulated after treatment with T-oligos.

Next, the researchers wanted to see if T-oligos would have an effect on metastases in vivo. The researchers pre-treated melanoma cells with T-oligos and injected them into mice. Forty days later, mice injected with pretreated cells had more than 90 percent fewer tumors than controls. Furthermore, the tumors they did have were much smaller than those seen in control animals. While some reduction of tumor size and volume can be seen in mice injected with control oligos, Gilchrest noted that the effect - possibly a consequence of DNA injection irrespective of sequence - was not statistically significant.

In a final experiment, the scientists investigated whether T-oligos could have an effect on already-established tumors. They first injected mice with melanoma cells to induce tumor formation. After tumors had been established, the researchers began a treatment regimen of twice-daily T-oligo or control injections. Three weeks later, animals treated with T-oligos had tumors that were less than one-fifth the size of control animals. Histological studies confirmed that tumor cells in the T-oligo-treated mice had undergone apoptosis, and that as in the cultured melanoma cells, markers of cell differentiation were being expressed.

Work to gauge the promise of T-oligos is ongoing. The technology was licensed to Delaware-based SemaCo. Inc., which has granted a sublicense to Carlsbad, Calif.-based CancerVax Corp. (See BioWorld Today, March 16, 2004).

Gilchrest said that the group has tested T-oligos in a variety of malignant cell lines, and "the beneficial effects of T-oligos are not restricted to melanoma; in everything we've looked at, they have similar effects." To date, tumor cell lines studied include breast, ovarian, pancreatic and squamous-cell carcinomas, fibrosarcoma, osteosarcoma and lymphoma.

One advantage of the T-oligo method is that it appears to affect malignant cells more strongly than normal ones. Current chemotherapy by and large targets all dividing cells, which accounts for its extreme toxicity, since there are many healthy cells that continue to divide.

In fact, one of the ironies of chemotherapy is that malignant cells can become increasingly resistant to chemotherapy, so chemotherapy at some point starts preferentially targeting normally dividing cells. In contrast, T-oligo's effects on normal cells appear to be transient, at least in the preclinical studies reported in FASEB.

"We hypothesize that a normal cell will use this signal as a sign to check itself out and then go back to business as usual," Gilchrest said, "whereas the T-oligos allow the malignant cell to recognize that it has all these problems."