Membrane Localization of Human Equilibrative Nucleoside Transporter 1 in Tumor Cells May Predict Response to Adjuvant Gemcitabine in Resected Cholangiocarcinoma Patients.

BACKGROUND: The use of gemcitabine as an adjuvant modality for cholangiocarcinoma (CC) is increasing, but limited data are available on predictive biomarkers of response. Human equilibrative nucleoside transporter 1 (hENT-1) is the major transporter involved in gemcitabine intracellular uptake. This study investigated the putative predictive role of hENT-1 localization in tumor cells of CC patients undergoing treatment with adjuvant gemcitabine. METHODS: Seventy-one consecutive patients with resected CC receiving adjuvant gemcitabine at our center were retrospectively analyzed by immunohistochemistry for hENT-1 localization in tumor cells. The main outcome measure was disease-free survival (DFS). Hazard ratios (HRs) of relapse and associated 95% confidence intervals (CIs) were obtained from proportional hazards regression models stratified on quintiles of propensity score. RESULTS: Twenty-three (32.4%) cases were negative for hENT-1, 22 (31.0%) were positive in the cytoplasm only, and 26 (36.6%) showed concomitant cytoplasm/membrane staining. Patients with membrane hENT-1 had a longer DFS (HR 0.49, 95% CI 0.24-0.99, p = .046) than those who were negative or positive only in the cytoplasm of tumor cells. Notably, the association between DFS and membrane hENT-1 was dependent on the number of gemcitabine cycles (one to two cycles: HR 0.96, 95% CI 0.34-2.68; three to four cycles: HR 0.99, 95% CI 0.34-2.90; five to six cycles: HR 0.27, 95% CI 0.10-0.77). CONCLUSION: hENT-1 localization on tumor cell membrane may predict response to adjuvant gemcitabine in CC patients receiving more than four cycles of chemotherapy. Further prospective randomized trials on larger populations are required to confirm these preliminary results, so that optimal gemcitabine-based chemotherapy may be tailored for CC patients in the adjuvant setting. IMPLICATIONS FOR PRACTICE: Gemcitabine is becoming an increasingly used adjuvant modality in cholangiocarcinoma (CC), but limited data are available on predictive biomarkers of response. In this study, patients receiving more than four cycles of adjuvant gemcitabine and harboring Human equilibrative nucleoside transporter 1 (hENT-1, the major transporter involved in gemcitabine intracellular uptake) on tumor cell membrane had a longer disease-free survival compared with patients negative or positive for hENT-1 only in the cytoplasm of tumor cells. Overall these results may lay the basis for further prospective randomized trials based on a larger population of patients and may prove useful for tailoring appropriate gemcitabine-based chemotherapy for CC patients in the adjuvant setting.

Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains.

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.

Selection and analysis of spontaneous reciprocal mitotic cross-overs in Saccharomyces cerevisiae.

We developed a system that allows the selection of the reciprocal products resulting from spontaneous mitotic cross-overs in the yeast Saccharomyces cerevisiae. A number of other types of genetic events, including chromosome loss, can be monitored with this system. For a 120-kb chromosome interval on chromosome V (CEN5-CAN1), the rate of mitotic cross-overs was 4x10(-5) per division, a rate approximately 25,000-fold lower than the meiotic rate of cross-overs. We found no suppression of mitotic cross-overs near the centromere of chromosome V, unlike the suppression observed for meiotic exchanges. The rate of reciprocal cross-overs was substantially (38-fold) elevated by treatment of cells with hydroxyurea, a drug that reduces nucleotide pools and slows DNA replication.