Combinatorial antileukemic disruption of oxidative homeostasis and mitochondrial stability by the redox reactive thalidomide 2-(2,4-difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) and flavopiridol.

2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) is a member of a recently identified class of redox-reactive thalidomide analogs that show selective killing of leukemic cells by increasing intracellular reactive oxygen species (ROS) and targeting multiple transcriptional pathways. Flavopiridol is a semisynthetic flavonoid that inhibits cyclin-dependent kinases and also shows selective lethality against leukemic cells. The purpose of this study is to explore the efficacy and mechanism of action of the combinatorial use of the redox-reactive thalidomide CPS49 and the cyclin-dependent kinase inhibitor flavopiridol as a selective antileukemic therapeutic strategy. In combination, CPS49 and flavopiridol were found to induce selective cytotoxicity associated with mitochondrial dysfunction and elevations of ROS in leukemic cells ranging from additive to synergistic activity at low micromolar concentrations. Highest synergy was observed at the level of ROS generation with a strong correlation between cell-specific cytotoxicity and reciprocal coupling of drug-induced ROS elevation with glutathione depletion. Examination of the transcriptional targeting of CPS49 and flavopiridol combinations reveals that the drugs act in concert to initiate a cell specific transcriptional program that manipulates nuclear factor-kappaB (NF-kappaB), E2F-1, and p73 activity to promote enhanced mitochondrial instability by simultaneously elevating the expression of the proapoptotic factors BAX, BAD, p73, and PUMA while depressing expression of the antiapoptotic genes MCL1, XIAP, BCL-xL, SURVIVIN, and MDM2. The coadministration of CPS49 and flavopiridol acts through coordinate targeting of transcriptional pathways that enforce selective mitochondrial dysfunction and ROS elevation and is therefore a promising new therapeutic combination that warrants further preclinical exploration.

Keratinocyte growth conditions modulate telomerase expression, senescence, and immortalization by human papillomavirus type 16 E6 and E7 oncogenes.

Keratinocytes undergo a finite number of divisions in culture before senescing. The high-risk human papillomavirus (HPV) E6 and E7 oncoproteins prevent keratinocyte senescence and extend life span by interacting with p53 and pRb, respectively, and also by transcriptionally activating the human telomerase reverse transcriptase (hTERT) gene, which encodes the catalytic subunit of telomerase. We correlated telomerase activity, which was measured by a highly sensitive and quantitative real-time quantitative-PCR-based telomeric repeat amplification protocol assay, with telomere length and the expression of hTERT, p16(INK4a), and HPV-16 E6 and E7 in keratinocytes grown under two culture conditions. Primary human foreskin keratinocytes (HFKs) cultured in keratinocyte serum-free medium on plastic senesced at approximately 13 population doublings (PDs). Senescence was accompanied by a dramatic increase in p16(INK4A) levels, a marked decrease in telomerase, and only a slight decrease in telomere length. In contrast, HFKs grown in F medium on 3T3 fibroblast feeders maintained elevated telomerase and lower levels of p16(INK4A) for 60 PDs before senescing approximately 81 PDs. E7 was shown to act synergistically with E6 to super induce telomerase expression in a feeder environment-dependent manner. Culture of both HFKs and HFK/16E6E7 cells in the feeder environment significantly increased the number of doublings that these cells could undergo without a significant reduction in telomere length. Finally, transfer of either HFKs or HFK/16E6E7 cells from plastic to the feeder fibroblast culture system significantly induced telomerase activity. This induction in telomerase was fully reversible and largely attributable to the medium. Our results suggest that the influence of keratinocyte culture conditions on the expression of telomerase and p16(INK4A) and on telomere maintenance is responsible, at least partially, for the differences in proliferative capacity, senescence, and HPV-keratinocyte interactions seen in the two culture systems.