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

Abstract

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 paraffinembedded biopsies has been a bottleneck to undertake highthroughput 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.

Equilibrative nucleoside transporter-2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells

“…As recently reported 19 these antibodies specifically recognized single bands of 50-55 kDa in CLL protein extracts. Figure 2 shows representative Western blots of five independent CLL samples in which hENT1 (a), hENT2 (b) and atubulin were analyzed.…”

“…19 They were raised against isoform-specific domains at the intracellular loop between transmembrane domains 6 and 7. Following incubations with these primary antibodies against hENT1 and hENT2, proteins were detected with the use of secondary antibodies conjugated to horseradish peroxidase and an enhanced chemiluminiscence (ECL) detection kit (Amersham, Buckinghamshire, UK).…”

“…18 Clinical correlations using the pattern of NT isoform expression have recently been obtained when monitoring NT occurrence in nearly 300 gynecological tumors using a tissue array approach. hENT1 and hENT2 expression was significantly retained in a high percentage of tumors, whereas hCNT1 expression was frequently reduced or lost, 19 particularly in some histotypes characterized by poor prognosis. Similarly, in a previous study using a small cohort of breast-cancer patients, only a few tumors were found to be negative for hENT1, 20 thus suggesting that, although hENT1 expression might show variability among tumors, a complete lack of hENT1 protein may be rare.…”

Identification of TIGAR in the equilibrative nucleoside transporter 2-mediated response to fludarabine in chronic lymphocytic leukemia cells

“…21 In fact, it has been reported that hENT1 expression, despite showing marked variability among individuals, is comparatively higher in most tumors compared to in their non-transformed tissue counterparts 38 and that hENT1 protein expression is strongly retained in gynecological tumors, particularly when compared to the highly efficient nucleoside-derived drug transporter protein hCNT1. 39 A general consensus in the field is that hENT1 is a ubiquitous, highly expressed transporter protein that can account for most of the nucleoside supply required for salvage processes, particularly during tissue growth. Uncontrolled proliferation processes do not appear to be implicated in the development of CLL and, in accordance with what has been discussed above, hENT1 expression contributes only slightly to fludarabineinduced cytotoxicity in CLL cells, 12,14 even though fludarabine is a hENT1 substrate.…”

Polymorphisms in ABCG2, ABCC3 and CNT1 genes and their possible impact on chemotherapy outcome of lung cancer patients

“…26,27 In some gynaecological tumors, the expression of CNT1 was found to be reduced, and this reduction of CNT1 protein was correlated with poor prognosis. 28 The SNP G565A results in reduced affinity for gemcitabine. 29 Response to chemotherapy will be influenced both by a patient's genetic background, i.e.…”