Short synthetic oligonucleotides (ODNs) can be used to block cellular processes involved in cell growth and proliferation. Often acting as aptamers, these molecules interact with critical proteins that regulate the induction of apoptosis or necrosis. We have used a specialized class of ODNs that contain a monomeric sequence of guanosine to induce apoptosis specifically in the malignant esophageal cell line, OE19, in cell culture, and in a NODscid mouse model. OE19 cells were grown in culture and treated with a stable G-rich oligonucleotide (GRO). Cells were processed and apoptosis was measured by FACS analyses, caspase activity, and Hoescht staining. Circular dichroism (CD) was used to define the structure and stability of various GROs. The GRO works by first inducing retardation in the progression of the cell cycle and then by creating a sub-G1 population of apoptotic cells. The reaction is dose dependent, and appears to rely on the capacity of the G-rich ODN to adopt a G-quartet conformation. Apoptosis was measured by determining caspase 3/7 levels and by staining for nuclear fragmentation using the Hoechst dye. Importantly, nonmalignant esophageal cells or normal human lung fibroblasts are not impeded in their cell cycle progression when incubated with the G-rich ODNs. These results suggest that a selective killing of esophageal tumor cells is directed by G-rich ODNs. Selective killing was demonstrated in the unique activity of the GRO compared to other ODNs of different sequences as well as the response of oncogenic cells compared to nononcogenic cells.

Toll-like receptors (TLR) are pattern recognition receptors that play a pivotal role in the initiation of immune responses. Here we report that the murine mammary carcinoma 4T1 constitutively expressed genes encoding TLR2, 3, 4 and 5. Moreover, treatment of the 4T1 cell line with peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly(I:C)) or lipopolysaccharide (LPS), agonists for TLR2, 3 or 4 respectively, induced nuclear translocation of NFkappaB and secretion of CCL2, CCL5 and CXCL1 in a dose dependent manner. Although treating the tumor cells with the TLR agonists did not modulate growth or viability of the tumor cells in vitro, 4T1 exhibited a decreased growth rate in vivo following treatment with LPS that was dependent upon the presence of CD8(+) T cells. Analysis of 3 additional murine mammary carcinomas revealed that they also secreted CCL2, CCL5 and CXCL1 in response to TLR agonist treatment, and LPS treated 168 and SM1 tumors exhibited decreased growth rates in vivo, but not in vitro. These data indicated that 4 out of 4 murine mammary carcinomas secreted proinflammatory chemokines following treatment with TLR agonists, and 3 out of 4 of the mammary carcinomas responded to LPS treatment in a manner that decreased tumor growth in vivo.

Gene repair employs single‐stranded DNA oligonucleotides (ODNs) to directly alter specific sequences. These ODNs, when transfected into mammalian cells, have been reported to stall the cells in S‐phase of the cell cycle. We have shown that a G‐rich oligonucleotide, G20, induces apoptosis in human esophageal cancer cells at greater than 72 hours. This effect seems to be unique to the G20 sequence; data collected using other sequences such as the A20, T20, and C20 ODNs do not produce the same apoptotic effects. We observe successful transfection by electroporation of the G‐rich oligonucleotide in several cell lines, as measured by FACS and confocal microscopy. We examined a variety of oligonucleotide sequences reported to form G‐quadruplexes, none of which result in the same effects as the G20 molecule. This demonstrates the uniqueness of the G20 in its initiation of the cellular response. In our efforts to determine the nature of this novel response, we examined the involvement of chromosomal telomeres; telomeric sequences are G‐rich in nature and form quadruplexes. To determine if the G20 ODN is mimicking a cellular telomeric sequence or interfering with proteins that interact with telomeres, we treated cells with a telomerase inhibitor, TMPyP4. While this inhibitor induces a minor G2/M stall, it does not correlate to the extent as the G20.

Toll-like receptors (TLR) are pattern recognition receptors that play a pivotal role in the initiation of immune responses. Ligation of TLR on antigen presenting cells enhances immunological responses through multiple mechanisms including secretion of proinflammatory mediators. Here we report that the murine mammary carcinoma 4T1 constitutively expressed genes encoding TLR2, 3, 4 and 5. Moreover, treatment of 4T1 with peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly(I:C)) or lipopolysaccharide (LPS), agonists for TLR2, 3 or 4 respectively, induced nuclear translocation of NFkB and secretion of CCL2, CCL5 and CXCL1 in a dose dependent manner. Although treating the tumor cells with the TLR agonists did not modulate growth or viability of the tumor cells in vitro, 4T1 exhibited a decreased growth rate in vivo following treatment with LPS that was dependent upon the presence of CD8+ T cells. Analysis of three additional murine mammary carcinomas revealed that they also secreted CCL2, CCL5 and CXCL1 in response to TLR agonist treatment, and LPS treated 168 and SM1 tumors exhibited decreased growth rates in vivo, but not in vitro. These data indicated that four out of four murine mammary carcinomas secreted proinflammatory chemokines following treatment with TLR agonists, and three out of four of the mammary carcinomas responded to LPS treatment in a manner that decreased tumor growth in vivo.