Disrupted plasma membrane localization of equilibrative nucleoside transporter 2 in the chemoresistance of human pancreatic cells to gemcitabine (dFdCyd).

Although the nucleoside pyrimidine analogue gemcitabine is the most effective single agent in the palliation of advanced pancreatic cancer, cellular resistance to gemcitabine treatment is a major problem in the clinical scene. To clarify the molecular mechanisms responsible for chemoresistance to gemcitabine, mRNA expression of the key enzymes including cytidine deaminase (CDA), deoxycytidine kinase (dCK), 5'-nucleotidase (NT5), equilibrative nucleoside transporter 1 and 2 (ENT1 and ENT2), dCMP deaminase (dCMPK), ribonucleotide reductase M1 and M2 (RRM1 and RRM2), thymidylate synthase (TS) and CTP synthase (CTPS) was examined. The interacellular uptake of gemcitabine was greatly impaired in the chemoresistant cell lines due to dysfunction of ENT1 and ENT2. Protein expression of ENT1 and ENT2 and their protein coding sequences were not altered. Immunohistochemical and western blot analyses revealed that localization of ENT2 on the plasma membrane was disrupted. These data suggest that the disrupted localization of ENT2 is one of causes of the impaired uptake of gemcitabine, resulting in a gain of chemoresistance to gemcitabine.

Systematic evaluation of a novel model for cardiac ischemic preconditioning in mice.

Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of transgenic mice seems critical. Due to technical difficulty associated with performing cardiac IP in mice, we developed an in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion. This technique has the major advantage of eliminating the necessity of intermittently occluding the coronary artery with a knotted suture. To systematically evaluate this model, we first demonstrated correlation of ischemia times (10-60 min) with infarct sizes [3.5 +/- 1.3 to 42 +/- 5.2% area at risk (AAR), Evan's blue/triphenyltetrazolium chloride staining]. IP (4 x 5 min) and cold ischemia (27 degrees C) reduced infarct size by 69 +/- 6.7% and 84 +/- 4.2%, respectively (n = 6, P < 0.01). In contrast, lower numbers of IP cycles did not alter infarct size. However, infarct sizes were distinctively different in mice from different genetic backgrounds. In addition to infarct staining, we tested cardiac troponin I (cTnI) as marker of myocardial infarction in this model. In fact, plasma levels of cTnI were significantly lower in IP-treated mice and closely correlated with infarct sizes (R(2) = 0.8). To demonstrate transcriptional consequences of cardiac IP, we isolated total RNA from the AAR and showed repression of the equilibrative nucleoside transporters 1-4 by IP in this model. Taken together, this study demonstrates highly reproducible infarct sizes and cardiac protection by IP, thus minimizing the variability associated with knot-based coronary occlusion models. Further studies on cardiac IP using transgenic mice may consider this technique.

Expression of the nucleoside-derived drug transporters hCNT1, hENT1 and hENT2 in gynecologic tumors.

Deoxynucleoside analogs are used in the treatment of a variety of solid tumors. Their transport across the plasma membrane may determine their cytotoxicity and thus nucleoside transporter (NT) expression patterns may be of clinical relevance. Lack of appropriate antibodies for use in paraffin-embedded biopsies has been a bottleneck to undertake high-throughput analysis of NT expression in solid tumors. Here we report the characterization of 2 new antibodies raised against the low-affinity equilibrative NTs, hENT1 and hENT2, suitable for that purpose. These 2 antisera, along with a previously characterized antibody that specifically recognizes the high-affinity Na-dependent concentrative NT, hCNT1, have been used to analyze, using a tissue array approach, NT expression in gynecologic cancers (90 ovarian, 80 endometrial and 118 uterine cervix carcinomas). Human CNT1 was not detected in 33% and 39% of the ovarian and uterine cervix carcinomas, respectively, whereas hENT1 and hENT2 expression was significantly retained in a high percentage of tumors (91% and 96% for hENT1, 84% and 98% for hENT2, in ovarian and cervix carcinomas, respectively). Only a few endometrial carcinomas (15%) were found to be negative for hCNT1, but they all retained hENT1 and hENT2 expression. In ovarian cancer, the loss of all 3 NT proteins was a more common event in the clear cell histologic subtype than in the serous, mucinous and endometrioid histotypes. In uterine cervix tumors, the loss of expression of hCNT1 was significantly associated with the adenocarcinoma subtype. In summary, hCNT1 was by far the isoform whose expression was most frequently reduced or lost in the 3 types of gynecologic tumors analyzed. Moreover, NT expression is related to the type of gynecologic tumor and its specific subtype, hCNT1 protein loss being highly correlated with poor prognosis histotypes. Since hCNT1, hENT1 and hENT2 recognize fluoropyrimidines as substrates, but with different affinities, this study anticipates high variability in drug uptake efficiency in solid tumors.