Julie K. Schwarz

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.

Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. Here we show that the ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and that T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells.

CONTEXT: Retrospective studies have demonstrated that the use of positron emission tomography (PET) with F-18 fluorodeoxyglucose (FDG) in the posttherapy evaluation of patients with cervical carcinoma is predictive of survival outcome. OBJECTIVE: To validate the association between the metabolic response on the 3-month posttherapy FDG-PET and long-term survival outcome. DESIGN, SETTING, AND PATIENTS: A prospective cohort study designed to validate our previous finding that the results of a 3-month posttherapy FDG-PET are predictive of long-term clinical outcome. A total of 92 women were treated with external irradiation, brachytherapy, and concurrent chemotherapy from January 2003 through September 2006. Posttherapy whole-body FDG-PET was performed 2 to 4 months (mean, 3 months) after completion of therapy. MAIN OUTCOME MEASURES: The primary outcome end points were metabolic response, progression-free survival, and cause-specific survival. RESULTS: Posttherapy FDG-PET showed a complete metabolic response in 65 patients (70%), a partial metabolic response in 15 (16%), and progressive disease in 12 (13%). Their 3-year progression-free survival rates were 78%, 33%, and 0%, respectively (P < .001). Multivariate analysis demonstrated that the hazard ratio (HR) for risk of recurrence based on the posttherapy metabolic response showing progressive disease was 32.57 (95% confidence interval [CI], 10.22-103.82). A partial metabolic response had an HR of 6.30 (95% CI, 2.73-14.56). These were more predictive of survival outcome than the pretreatment lymph node status (HR, 3.54; 95% CI, 1.54-8.09). CONCLUSION: In this single-site study population of women with cervical cancer, 3-month posttherapy FDG uptake, as detected by whole-body PET, was predictive of survival.

Invasive cervical cancer is a leading cause of cancer death in women worldwide, resulting in about 300,000 deaths each year. The clinical outcomes of cervical cancer vary significantly and are difficult to predict. Thus, a method to reliably predict disease outcome would be important for individualized therapy by identifying patients with high risk of treatment failures before therapy. In this study, we have identified a microRNA (miRNA)-based signature for the prediction of cervical cancer survival. miRNAs are a newly identified family of small noncoding RNAs that are extensively involved in human cancers. Using an established PCR-based miRNA assay to analyze 102 cervical cancer samples, we identified miR-200a and miR-9 as two miRNAs that could predict patient survival. A logistic regression model was developed based on these two miRNAs and the prognostic value of the model was subsequently validated with independent cervical cancers. Furthermore, functional studies were done to characterize the effect of miRNAs in cervical cancer cells. Our results suggest that both miR-200a and miR-9 could play important regulatory roles in cervical cancer control. In particular, miR-200a is likely to affect the metastatic potential of cervical cancer cells by coordinate suppression of multiple genes controlling cell motility.