To understand the mechanisms controlling platelet-derived growth factor receptor β (PDGFR-β) expression in malignancies, we have cloned and characterized the first functional promoter of the human PDGFR-β gene, which has been confirmed by luciferase reporter gene assays. The transcription initiation sites were mapped by primer extension. Promoter deletion experiments demonstrate that the proximal, highly GC-rich region (−165 to −139) of the human PDGFR-β promoter is crucial for basal promoter activity. This region is sensitive to S1 nuclease and likely to assume a non-B-form DNA secondary structure within supercoiled plasmid. The G-rich strand in this region contains a series of runs of three or more guanines that can form multiple different Gquadruplex structures, which have been subsequently assessed by circular dichroism (CD). A Taq polymerase stop assay has shown that three different G-quadruplex-interactive drugs can each selectively stabilize different G-quadruplex structures of the human PDGFR-β promoter. However, in transfection experiments only telomestatin significantly reduced the human PDGFR-β basal promoter activity relative to the control. Furthermore, the PDGFR-β mRNA level in Daoy cells was significantly decreased after treatment with 1 μM telomestatin for 24 hours. Therefore we propose that ligand-mediated stabilization of specific G-quadruplex structures in the human PDGFR-β promoter can modulate its transcription.

Topoisomerase inhibitors are important and clinically effective drugs, while G-quadruplex-interactive compounds that disrupt telomere maintenance mechanisms have yet to be proven useful in the clinic. If G-quadruplex-interactive compounds are to be clinically useful, it will most likely be in combination with more established cytotoxic agents. We have previously reported on a family of topoisomerase II inhibitors that also interact with G-quadruplexes. On the basis of previously established structure-activity relationships (SARs) for compounds that are able to inhibit topoisomerase II or interact with G-quadruplex to varying degrees, we have now designed and synthesized four new fluoroquinoanthroxazines (FQAs) that have different profiles of mixed topoisomerase II poisoning effects and G-quadruplex interactions. The biological profiles of the four new compounds were determined with respect to G-quadruplex interaction (polymerase stop and photocleavage assays) and topoisomerase II interaction (DNA cleavage and kDNA decatenation assays), alongside cytotoxicity tests with matched pairs of topoisomerase II-resistant and topoisomerase II-sensitive cells and with telomerase (+) and ALT (+) cell lines (ALT = alternative lengthening of telomeres). From this study, we have identified two FQAs with sharply contrasting profiles of potent G-quadruplex interaction with a weak topoisomerase II poisoning effect, and vice versa, for further evaluation to determine the optimum combination of these activities in subsequent in vivo studies.

We have demonstrated that a parallel G-quadruplex structure in the c-MYC promoter functions as a transcriptional repressor element. Furthermore, a specific G-to-A mutation in this element results in destabilization of the G-quadruplex repressor element and an increase in basal transcriptional activity. To validate this model in an in vivo context, we have examined the sequence of this region in human colorectal tumors and the surrounding normal tissue. We have found that ≈30% of tumors contain one of two specific G-to-A mutations, not present in the surrounding normal tissue, that destabilize the parallel G-quadruplex, which would be expected to give rise to abnormally high expression of c-MYC in these cells. In contrast, G-quadruplex-disruptive mutations were absent in 20 colon adenomas, suggesting that these mutations occur late in tumorigenesis. We have also demonstrated that these same mutations are found in established colorectal cell lines. NM23-H2 levels are lower in cancer tissues and cell lines that harbor these mutations. In cells with repressed levels of NM23-H2, the mutated and destabilized G-quadruplex silencer element can be reinstated by the addition of G-quadruplex-stabilizing compounds, providing an opportunity for therapeutic intervention for patients carrying these mutations

Recent evidence has supported the hypothesis that chemotherapeutic drugs and radiation induce an apoptotic pathway that requires the active participation of the cell. One pathway of apoptosis in malignant lymphoid cells is mediated by the Fas antigen. We studied the human myeloma (8226) and T-cell leukemia (CEM) cell lines selected for resistance to the anthracenes, doxorubicin or mitoxantrone, by continuous culture in the presence of either agent. We found that these drug-resistant cell lines were also resistant to Fas-mediated apoptosis in a dose-dependent manner. The degree of resistance to Fas-mediated apoptosis correlated directly with the level of resistance to chemotherapeutic drugs. These observations indicate that, as cancer cell lines develop mechanisms of drug resistance, they may also develop mechanisms of resistance to physiologic signals of apoptosis. Two mechanisms of resistance to Fas-mediated apoptosis were observed in these cell lines. One mechanism was associated with a dose-dependent reduction in the surface expression of Fas antigen. Analysis of RNA by reverse transcriptase-polymerase chain reaction assays showed that the reduction of Fas antigen expression occurred at the level of transcription. A second mechanism of drug resistance showed no decrease of Fas antigen expression; however, the apoptotic response was diminished. In this situation, removal of the chemotherapeutic agent resulted in a partial reversion to chemosensitivity and re-expression of the Fas antigen, but these cell lines did not regain the ability to undergo apoptosis in response to cross-linking by anti-Fas antibody. These findings support the hypothesis that apoptosis mediated by both chemotherapeutic agents and physiologic stimuli may share a common downstream effector. The demonstration that selection for drug resistance in hematopoietic cell lines results in a simultaneous resistance to Fas-mediated apoptosis may have clinical implications in the development of strategies for the treatment of resistant disease. Further analysis of the molecular mechanisms of Fas expression and function will facilitate the design of biological response modifying agents for the treatment of malignancy.