Depletion of cyclin E in breast cancer cells by treatment with the transcription inhibitor, 8-chloro-adenosine
Authors: Christine Stellrecht, Rishi Arya, Varsha Gandhi
Journal: Cancer Research (2007)

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA

3274 

Cyclin E expression is dysregulated in many types of cancer with overexpression in all phases of the cell cycle. In breast cancer, high levels of cyclin E, particularly its hyperactive low molecular weight (LMW) isoforms, have been associated with a poor prognosis and an aggressive phenotype. Thus, therapies targeting cyclin E should be beneficial for the treatment of breast cancer. In recent years, it has become apparent that RNA transcription is a potential target for cancer therapeutics as RNA directed agents exploit the tumor cells’ dependency on short-lived oncogenes, growth regulators, and survival factors. Because the half-life of cyclin E mRNA and protein is as short as 30 min, its expression is likely to be affected by transcription inhibitors. The nucleoside analog, 8-chloro-adenosine (8-Cl-Ado), is cytotoxic to a number of epithelial, hematological, and neuronal cell lines. Previously, we demonstrated that in hematopoeitic cells 8-Cl-Ado inhibits global RNA synthesis, with a maximum effect on mRNA production. This inhibition was due to analog incorporation into RNA causing premature termination [Cancer Res. 63:7968, 2003] and obstruction of poly(A) polymerase activity [JBC 279:40405, 2004]. Metabolic studies in the breast cancer cell lines, MCF-7 and BT-474, show that the triphosphate form of this analog rapidly accumulated to >100 μM by 3 h incubation with 10 μM 8-Cl-Ado. This accumulation was time dependent and reached > 200 and 300 μM by 12 h, respectively. Parallel to accumulation, there was a 50 - 70% decrease in the endogenous ATP pool. To determine the extent of RNA synthesis inhibition in these cell lines, [3H]-uridine incorporation was measured at various times of treatment. RNA synthesis was rapidly inhibited in 3 h to 60 and 40% of control, and by 12 h to 40 and 30% in MCF-7 and BT-474 cells, respectively. To determine if this decrease in RNA synthesis resulted in a depletion of cyclin E expression, real-time RT-PCR and immunoblot analyses were performed. The cyclin E mRNA rapidly decreased in the first 12 h of treatment and reached a maximal decrease of ~70% by 24 h. Concomitantly, there was a decrease in the protein levels of both full length and LMW cyclin E isoforms, demonstrating the efficacy of the analog in depleting cyclin E expression in breast cancer cells. Interestingly, during this time period there was no change in the cell cycle profile, except that by 2 days the sub-G1 population started to increase and was 7 and 35 fold higher by 4 days in BT-474 and MCF-7 cells, respectively. PARP cleavage, another apoptosis marker, was induced within 2 day of 8-Cl-Ado treatment in these cells. In conclusion, our data suggest 8-Cl-Ado inhibits transcription and induces apoptosis in the breast cancer cell lines MCF-7 and BT-474. This was associated with a reduced cyclin E expression, indicating that the cytotoxic activities may be mediated through the depletion of cyclin E expression.