The absence of human equilibrative nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine-treated pancreas adenocarcinoma.

PURPOSE: Gemcitabine monotherapy is the standard palliative chemotherapy for pancreatic adenocarcinoma. Gemcitabine requires plasma membrane nucleoside transporter proteins to efficiently enter cells and exert it cytotoxicity. In vitro studies have demonstrated that deficiency of human equilibrative nucleoside transporter 1 (hENT1), the most widely abundant and distributed nucleoside transporter in human cells, confers resistance to gemcitabine toxicity, but the distribution and abundance of nucleoside transporters in normal and malignant pancreatic tissue is unknown. EXPERIMENTAL DESIGN: We studied tumor blocks from normal pancreas and 21 Alberta patients with gemcitabine-treated pancreatic cancer. Immunohistochemistry on the formalin-fixed, paraffin-embedded tissues was performed with specific hENT1 and human Concentrative Nucleoside Transporter 3 monoclonal antibodies and scored by a pathologist blinded to clinical outcomes. RESULTS: hENT1 was detected in normal Langerhan cells and lymphocytes but not in normal glandular elements. Patients in whom all adenocarcinoma cells had detectable hENT1 had significantly longer median survivals from gemcitabine initiation than those for whom hENT1 was absent in a proportion (10 to 100%) of adenocarcinoma cells (median survival, 13 versus 4 months, P = 0.01). Immunohistochemistry for human Concentrative Nucleoside Transporter 3 revealed moderate to high-intensity staining in all adenocarcinoma tissue samples. CONCLUSIONS: Patients with pancreatic adenocarcinoma with uniformly detectable hENT1 immunostaining have a significantly longer survival after gemcitabine chemotherapy than tumors without detectable hENT1. Immunohistochemistry for hENT1 shows promise as a molecular predictive assay to appropriately select patients for palliative gemcitabine chemotherapy but requires formal validation in prospective, randomized trials.     

Human equilibrative nucleoside transporter 1 is associated with the chemosensitivity of gemcitabine in human pancreatic adenocarcinoma and biliary tract carcinoma cells

Gemcitabine has been one of the most commonly used agents for pancreatic adenocarcinoma chemotherapy, but the determinants of the sensitivity of and resistance to this agent are not yet fully understood. In this study with pancreatic carcinoma and biliary tract carcinoma cell lines, we examined the gene expression levels of nucleotide transporters and others related to the metabolism of gemcitabine in the light of sensitivity to this agent. Quantitative RT-PCR demonstrated that one of the nucleotide transporter genes; human equilibrative nucleoside transporter 1 (hENT1) was associated with the sensitivity to gemcitabine as represented by IC50, while the other genes for nucleotide transporter and metabolism were not. We conclude that increased hENT1 expression is a most important determinant of gemcitabine sensitivity at least in an in vitro study.     

Differential expression of human equilibrative nucleoside transporter 1 (hENT1) protein in the Reed-Sternberg cells of Hodgkin's disease

Gemcitabine is a cytotoxic nucleoside analog with activity in relapsing/refractory Hodgkin's disease (HD). Because gemcitabine is hydrophilic, it requires plasma membrane nucleoside transporter proteins to access intracellular targets. The most abundant and widely distributed transporter in human cells is human equilibrative nucleoside transporter 1 (hENT1). Because our prior studies showed that a deficiency in hENT1 confers high-level resistance to gemcitabine toxicity in vitro, we developed an immunohistochemical method to assess the hENT1 abundance of cells in tumor tissue. We now report the application of this method for visualizing the hENT1 protein abundance in the plasma membranes of Reed-Sternberg cells in lymph nodes of HD patients. Frozen sections of 30 lymph nodes were stained with monoclonal antibodies (mAb 10D7G2) raised against a synthetic peptide comprised of residues 254-271 from the large intracellular loop of hENT1 and staining intensity was scored on a 0-4 + scale. hENT1-staining intensity varied among HD lymph node samples (score/n; 0/8; 1/10; 2/9; 3/3; 4/0) and suggested that at least 60% of the tumors appeared hENT1 deficient. Because Epstein-Barr virus (EBV) is often associated with HD, staining for Epstein-Barr early RNA was also examined. Although 9/30 patients tested positive for EBV, there was no correlation with hENT1 staining. hENT1-staining intensities were positively correlated with age of the patient but were independent of other clinical, laboratory or pathology features (tumor stage, histologic subtype, presence of B symptoms, staining for CD15 or CD30, serum biochemistry, disease free survival, and overall survival). We conclude that, because hENT1 deficiency has been previously related to nucleoside-drug resistance, immunohistochemical staining for hENT1 warrants evaluation as a predictive tool for guiding the appropriate use of gemcitabine in the treatment of HD.     

Enhanced efficacy of gemcitabine by indole-3-carbinol in pancreatic cell lines: the role of human equilibrative nucleoside transporter 1

Pancreatic cancer patients treated with gemcitabine (2',2'-difluorodeoxycytidine) can eventually develop resistance. Recently, published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C). The current study examined the possible mechanism for this I3C-enhanced efficacy. Several pancreatic cell lines (BxPC-3, Mia Paca-2, PL-45, AsPC-1 and PANC-1) were examined for modulation of human equilibrative nucleoside transporter 1 (hENT1) expression, the major transporter for gemcitabine, by I3C alone and combined with gemcitabine. I3C significantly (p<0.01) up-regulated hENT1 expression in several cell lines. Gemcitabine alone showed no effect on hENT1 expression. However, combining gemcitabine with I3C further increased hENT1 expression. Cell viability assays revealed no effect of I3C on normal cells, hTERT-HPNE. hENT1-specific inhibitor, nitrobenzylthioinosine, significantly abrogated I3C-induced gemcitabine cytotoxicity, further demonstrating its specificity. This study demonstrates that up-regulation of hENT1 expression may be a novel mechanism involved in the additive effect of I3C and gemcitabine.     

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.     

CNT1 expression influences proliferation and chemosensitivity in drug-resistant pancreatic cancer cells

Overcoming the inherent chemoresistance of pancreatic cancers remains a major goal of therapeutic investigations in this disease. In this study, we discovered a role for the human concentrative nucleoside transporter-1 (hCNT1; SLC28A1), a high-affinity pyrimidine nucleoside transporter, in determining the chemosensitivity of human pancreatic cancer cells to gemcitabine, the drug used presently as a standard of care. Compared with normal pancreas and pancreatic ductal epithelial cells, hCNT1 expression was frequently reduced in pancreatic tumors and tumor cell lines. In addition, hCNT1-mediated (3)H-gemcitabine transport was lower in pancreatic cancer cell lines and correlated with cytotoxic IC(50) estimations of gemcitabine. In contrast to gemcitabine-sensitive pancreatic cancer cell lines, MIA PaCa-2, a gemcitabine-resistant pancreatic cancer cell line, exhibited relatively restrictive, cell cycle-dependent hCNT1 expression and transport. hCNT1 translation was suppressed in the late G1-enriched MIA PaCa-2 cell population possibly in an miRNA-dependent manner, which corresponded with the lowest hCNT1-mediated gemcitabine transport during this phase. Although hCNT1 protein was induced during G1/S transition, increased hCNT1 trafficking resulted in maximal cell surface recruitment and transport-overshoot in the G2/M phase-enriched cell population. hCNT1 protein was directed predominantly to proteasomal or lysosomal degradation in S or G2/M phase MIA PaCa-2 cells, respectively. Pharmacological inhibition of hCNT1 degradation moderately increased cell surface hCNT1 expression and cellular gemcitabine transport in MIA PaCa-2 cells. Constitutive hCNT1 expression reduced clonogenic survival of MIA PaCa-2 cells and steeply augmented gemcitabine transport and chemosensitization. In addition to supporting a putative tumor suppressor role for hCNT1, our findings identify hCNT1 as a potential candidate to render drug-resistant pancreatic cancer cells amenable to chemotherapy.     

Mechanisms of uptake and resistance to troxacitabine, a novel deoxycytidine nucleoside analogue, in human leukemic and solid tumor cell lines

Troxacitabine (Troxatyl; BCH-4556; (-)-2'-deoxy-3'-oxacytidine), a deoxycytidine analogue with an unusual dioxolane structure and nonnatural L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work was undertaken to identify potential biochemical mechanisms of resistance to troxacitabine and to determine whether there are differences in resistance mechanisms between troxacitabine, gemcitabine, and cytarabine in human leukemic and solid tumor cell lines. The CCRF-CEM leukemia cell line was highly sensitive to the antiproliferative effects of troxacitabine, gemcitabine, and cytarabine with inhibition of proliferation by 50% observed at 160, 20, and 10 nM, respectively, whereas a deoxycytidine kinase (dCK)-deficient variant (CEM/dCK(-)) was resistant to all three drugs. In contrast, a nucleoside transport-deficient variant (CEM/ARAC8C) exhibited high levels of resistance to cytarabine (1150-fold) and gemcitabine (432-fold) but only minimal resistance to troxacitabine (7-fold). Analysis of troxacitabine transportability by the five molecularly characterized human nucleoside transporters [human equilibrative nucleoside transporters 1 and 2, human concentrative nucleoside transporter (hCNT) 1, hCNT2, and hCNT3] revealed that short- and long-term uptake of 10-30 microM [(3)H]troxacitabine was low and unaffected by the presence of either nucleoside transport inhibitors or high concentrations of nonradioactive troxacitabine. These results, which suggested that the major route of cellular uptake of troxacitabine was passive diffusion, demonstrated that deficiencies in nucleoside transport were unlikely to impart resistance to troxacitabine. A troxacitabine-resistant prostate cancer subline (DU145(R); 6300-fold) that exhibited reduced uptake of troxacitabine was cross-resistant to both gemcitabine (350-fold) and cytarabine (300-fold). dCK activity toward deoxycytidine in DU145(R) cell lysates was <20% of that in DU145 cell lysates, and no activity was detected toward troxacitabine. Sequence analysis of cDNAs encoding dCK revealed a mutation of a highly conserved amino acid (Trp(92)-->Leu) in DU145(R) dCK, providing a possible explanation for the reduced phosphorylation of troxacitabine in DU145(R) lysates. Reduced deamination of deoxycytidine was also observed in DU145(R) relative to DU145 cells, and this may have contributed to the overall resistance phenotype. These results, which demonstrated a different resistance profile for troxacitabine, gemcitabine, and cytarabine, suggest that troxacitabine may have an advantage over gemcitabine and cytarabine in human malignancies that lack or have low nucleoside transport activities.     

Immunohistochemical variation of human equilibrative nucleoside transporter 1 protein in primary breast cancers.

Gemcitabine and capecitabine are nucleoside analogues used in chemotherapy strategies for the treatment of breast cancer. We previously demonstrated that deficiency in hENT1, the most abundant and widely distributed plasma membrane nucleoside transporter in human cells, confers high-level resistance to gemcitabine toxicity in vitro, whereas the relationship between hENT1 activity and capecitabine toxicity is unknown. To determine the relationship between capecitabine cytotoxicity and hENT1 abundance, cultured MDA-MB-435s human mammary carcinoma cells were exposed to graded concentrations of the capecitabine metabolites, 5'-deoxy-5-fluorouridine or 5-fluorouracil, in the presence and absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), a tight-binding inhibitor of hENT1. The presence of NBMPR reduced the cytotoxic effects of 5'-deoxy-5-fluorouridine, indicating that hENT1 also enabled cellular uptake of this capecitabine metabolite by breast cancer cells. We report here the development of an immunohistochemical method to assess the hENT1 abundance of malignant cells in solid tumors. Frozen sections of 33 primary breast cancers were stained with monoclonal antibodies raised against a synthetic peptide derived from the large intracellular loop of hENT1, and staining intensity was scored on a 0-4+ scale. hENT1 staining intensity varied markedly among breast samples (4 with score 0, 5 with score 1+, 7 with score 2+, 14 with score 3+, 3 with score 4+), suggesting that at least 9 of the tumors were hENT1 deficient. We conclude that because hENT1 deficiency has previously been associated with nucleoside drug resistance, immunohistochemical staining of hENT1 warrants further study as a predictive tool for guiding the appropriate use of gemcitabine and capecitabine in the treatment of breast cancer.     

cN-II expression predicts survival in patients receiving gemcitabine for advanced non-small cell lung cancer

Resistance to gemcitabine is likely to be multifactorial and could involve a number of mechanisms involved in drug penetration, metabolism and targeting. In vitro studies of resistant human cell lines have confirmed that human equilibrative nucleoside transporter 1 (hENT1)-deficient cells display resistance to gemcitabine. Overexpression of certain nucleotidases, such as cN-II, has also been frequently shown in gemcitabine-resistant models. In this study, we applied immunohistochemical methods to assess the protein abundance of cN-II, hENT1, human concentrative nucleoside transporter 3 (hCNT3) and deoxycitidine kinase (dCK) in malignant cells in from 43 patients with treatment-na?ve locally advanced or metastatic non-small cell lung cancer (NSCLC). All patients subsequently received gemcitabine-based chemotherapy. Response to chemotherapy, progression-free survival (PFS), and overall survival (OS) were correlated with abundance of these proteins. Among the 43 samples, only 7 (16%) expressed detectable hENT1, with a low percentage of positive cells, 18 expressed hCNT3 (42%), 36 (86%) expressed cN-II and 28 (66%) expressed dCK. In univariate analysis, only cN-II expression levels were correlated with overall survival. None of the parameters were correlated with freedom from progression survival nor with response. Patients with low levels of expression of cN-II (less than 40% positively stained cells) had worse overall survival than patients with higher levels of cN-II expression (6 months and 11 months, respectively). In a multivariate analysis taking into account age, sex, weight loss, stage and immunohistochemical results, cN-II was the only predictive factor associated with overall survival. This study suggests that cN-II nucleotidase expression levels identify subgroups of NSCLC patients with different outcomes under gemcitabine-based therapy. Larger prospective studies are warranted to confirm the predictive value of cN-II in these patients.     

Determinants of sensitivity and resistance to gemcitabine: the roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non-small cell lung cancer

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT-PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non-small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r =-0.6769, P = 0.0005), whereas dCK expression levels were not. In a highly gemcitabine-sensitive cell line, NCI-H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI-H23 cells to gemcitabine and subsequently established the drug-resistant clone H23/GEM-R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.     

Nucleoside transporter expression and activity is regulated during granulocytic differentiation of NB4 cells in response to all-trans-retinoic acid

NB4 cells express multiple nucleoside transporters (NTs), including: hENT1 (es), and hENT2 (ei), and the CNT subtype referred to as, csg; a concentrative sensitive guanosine specific transporter. csg activity is a distinguishing feature of the NB4 cell line and its presence suggests a particular requirement of these cells for guanosine salvage. Proliferation and differentiation pathways determine, in part, the number of NTs in cells and tissues. In this study, all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of NB4 cells resulted in biphasic changes in guanosine transport. Transient increases in csg and es activity, the result of an increase in V(max) (pmol/muls) of both transporter systems, served as early markers of differentiation while expression of a fully differentiated phenotype was accompanied by a selective loss of csg activity and the return of es activity to that of proliferating cells. Intracellular incorporation of [(3)H]-guanosine decreased as cells matured despite increased transport rates and suggested a reduced intracellular requirement of NB4-granulocytes compared to their proliferating counterparts. Whether a loss of csg activity could serve to assess clinical response to differentiation therapies is not known. Nitrobenzylthioinosine (NBMPR) binding sites within nuclear membrane (NM) preparations, suggested the presence of functional intracellular NTs. An increase in plasma membrane (PM) associated transporters coincided with the early increase in guanosine transport and a decrease in NBMPR binding to NM fractions and suggests that intracellular NTs may serve as a reserve pool for translocation to the (PM) when additional transport capacity is required. The modulation of transporters during differentiation could potentially regulate drug bioavailability and cytotoxicity and should be evaluated prior to combining differentiating agents with traditional nucleoside analogs in the treatment of APL.     

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

Fludarabine is considered the treatment of choice for most patients with chronic lymphocytic leukemia (CLL). We have analyzed the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in CLL cells. Among the known plasma membrane transporters, we have previously observed a significant correlation between fludarabine uptake via ENT carriers and ex vivo sensitivity of CLL cells to fludarabine, although mRNA amounts of the equilibrative nucleoside transporters hENT1 and hENT2 do not show any predictive response to treatment. In this study, using polyclonal monospecific antibodies we have observed a significant correlation between the expression of hENT2 by Western blot and fludarabine uptake via hENT carriers and also with ex vivo sensitivity of CLL cells to fludarabine. These results suggest that the equilibrative nucleoside transporter hENT2 plays a role in fludarabine responsiveness in CLL patients.     

Human equilibrative nucleoside transporter 1 (hENT1) protein is associated with short survival in resected ampullary cancer.

BACKGROUND: Gemcitabine is an acceptable alternative to best supportive care in the treatment of advanced biliary tract cancers. The human equilibrative nucleoside transporter 1 (hENT1) is a ubiquitous protein and is the major means by which gemcitabine enters human cells. Moreover, recent reports indicate a significant correlation between immunohistochemical variations of hENT1 in tumor samples and survival after gemcitabine therapy in patients with solid tumors. Materials and methods: We used immunohistochemistry to assess the abundance and distribution of hENT1 in tumor samples from radically resected cancer of the ampulla, and sought correlations between immunohistochemical results and clinical parameters including disease outcomes. RESULTS: In the 41 individual tumors studied, 12 (29.3%) had uniformly high hENT1 immunostaining. Statistical analysis showed a significant correlation between hENT1 and Ki-67 (P = 0.04). No statistical significant differences were found between immunohistochemical findings and patient characteristics (sex, age, and tumor-node-metastasis). On univariate analysis, hENT1 and Ki-67 expression were associated with overall survival (OS). Specifically, those patients with overexpression of hENT1 showed a shorter OS (P = 0.022) and those with high Ki-67 staining showed a shorter survival (P = 0.05). CONCLUSIONS: hENT1 expression is a molecular prognostic marker for patients with resected ampullary cancer and holds promise as a predictive factor to assist in chemotherapy decisions.     

Novel leukemic cell lines resistant to clofarabine by mechanisms of decreased active metabolite and increased antiapoptosis

Clofarabine (CAFdA) is incorporated into leukemic cells by human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. CAFdA is then phosphorylated to the active metabolite CAFdA triphosphate (CAFdATP) by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Two novel CAFdA‐resistant variants were established and their mechanism of resistance was elucidated. The two variants (HL/CAFdA20, HL/CAFdA80) were 20‐fold and 80‐fold more CAFdA‐resistant than HL‐60, respectively. mRNA levels of hENT1, hENT2 and hCNT3 were 53.9, 41.8 and 17.7% in HL/CAFdA20, and 30.8, 13.9 and 7.9% in HL/CAFdA80, respectively, compared with HL‐60. Thus, the total nucleoside transport capacity of CAFdA was reduced in both variants. dCK protein levels were 1/2 in HL/CAFdA20 and 1/8 in HL/CAFdA80 of that of HL‐60. dGK protein levels were 1/2 and 1/3, respectively. CAFdATP production after 4‐h incubation with 10 μM CAFdA was 20 pmol/10⁷cells in HL/CAFdA20 and 3 pmol/10⁷cells in HL/CAFdA80 compared with 63 pmol/10⁷cells in HL‐60. The decreased CAFdATP production attenuated drug incorporation into both mitochondrial and nuclear DNA. In addition, the two variants were resistant to CAFdA‐induced apoptosis due to Bcl2 overexpression and decreased Bim. A Bcl2 inhibitor, ABT737, acted synergistically with CAFdA to inhibit the growth with combination index values of 0.27 in HL/CAFdA20 and 0.23 in HL/CAFdA80, compared with 0.65 in HL‐60. Thus, the mechanism of resistance primarily included not only reduced CAFdATP production, but also increased antiapoptosis. The combination of CAFdA and ABT737 may be effective against CAFdA resistance.     

Expression of nucleoside transporters, deoxycitidine kinase, ribonucleotide reductase regulatory subunits, and gemcitabine catabolic enzymes in primary ovarian cancer

Purpose

Gemcitabine (2′,2′-difluorodeoxycytidine) (GEM) is one of the most actively investigated drugs in ovarian cancer. Many molecular mechanisms have been proposed to be involved in GEM sensitivity/resistance including the equilibrative nucleoside transporter-1 (hENT1), the concentrative nucleoside transporter-1 (hCNT1), and deoxycytidine kinase (dCK). The expression of the ribonucleotide reductase regulatory subunits M1 (RRM1) and M2 (RRM2), and the catabolic enzymes 5′nucleotidase (5′NT), and cytidine deaminase (CDA) play also an important role. The aim of the study is to investigate the potential clinical role of hENT1, hCNT1, dCK, RRM1, RRM2, CDA, and 5′NT in a single institutional series of 25 primary ovarian carcinomas.

Methods

The expression levels of the target genes were assayed by means of real-time quantitative PCR. The clinical role of the expression levels of each parameter was analyzed by Kaplan and Meier method and the Cox’s proportional hazards model.

Results

hENT1, hCNT1, dCK, CDA, 5′NT, RRM1, and RRM2 gene expression was documented in all samples; undifferentiated and clear cell carcinoma exhibited higher levels of hENT1, dCK, 5′NT, and RRM1 compared to serous ovarian tumors. As of May 2009, the median follow-up was 32 months (range 10–80), and death of disease were observed in 17 cases (68.0%). A statistically significant direct association of RRM2 expression levels and the relative risk of death was observed (Χ ² = 8.18, P = 0.0043). Moreover, cases with high RRM2 expression had a shorter OS (median OS = 19 months) than cases with low RRM2 levels (median OS = 36 months) (P = 0.0506).

Conclusions

We first reported that the most relevant genes involved in gemcitabine metabolism are expressed in ovarian carcinoma, and might be associated with more aggressive histotypes. The assessment of the expression levels of RRM2 as marker of clinical outcome deserves further investigation in a larger series of ovarian cancer patients.     

Prolonged versus standard gemcitabine infusion: translation of molecular pharmacology to new treatment strategy

Gemcitabine is frequently used in the treatment of patients with solid tumors. Gemcitabine is taken up into the cell via human nucleoside transporters (hNTs) and is intracellularly phosphorylated by deoxycytidine kinase (dCK) to its monophosphate and subsequently into its main active triphosphate metabolite 2',2'-difluorodeoxycytidine triphosphate (dFdCTP), which is incorporated into DNA and inhibits DNA synthesis. In addition, gemcitabine is extensively deaminated to 2',2'-difluorodeoxyuridine, which is largely excreted into the urine. High expression levels of human equilibrative nucleoside transporter type 1 were associated with a significantly longer overall survival duration after gemcitabine treatment in patients with pancreatic cancer. Clinical studies in blood mononuclear and leukemic cells demonstrated that a lower infusion rate of gemcitabine was associated with higher intracellular dFdCTP levels. Prolonged infusion of gemcitabine at a fixed dose rate (FDR) of 10 mg/m2 per minute was associated with a higher intracellular accumulation of dFdCTP, greater toxicity, and a higher response rate than with the standard 30-minute infusion of gemcitabine in patients with pancreatic cancer. In the current review, we discuss the molecular pharmacology of nucleoside analogues and the influence of hNTs and dCK on the activity and toxicity of gemcitabine, which is the basis for clinical studies on FDR administration, and the results of FDR gemcitabine administration in patients. These findings might aid optimal clinical application of gemcitabine in the future.     

Roles for hENT1 and dCK in gemcitabine sensitivity and malignancy of meningioma

BackgroundHigh-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors, and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown.MethodsWe examined the roles of hENT1 (human equilibrative nucleoside transporter 1) and dCK (deoxycytidine kinase) in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity.ResultshENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients.ConclusionsThe present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine.     

Expression of the human concentrative nucleotide transporter 1 (hCNT1) gene correlates with clinical response in patients affected by Waldenström's Macroglobulinemia (WM) and small lymphocytic lymphoma (SLL) undergoing a combination treatment with 2-chloro-2′-deoxyadenosine (2-CdA) and Rituximab

PurposeResistance to nucleoside analogues agents is likely to be multifactorial and could involve a number of mechanisms affecting drug penetration, metabolism and targeting. In vitro studies of resistant human cell lines have confirmed that human concentrative nucleoside transporter 1 (hCNT1)-deficient cells display resistance.Experimental designWe applied real-time PCR method to assess the mRNA expression of equilibrative and concentrative nucleoside transporter (hENT1, hCNT1), deoxycytidine and deoxyguanosine kinase (dCK, dGK), 5′-nucleotidase (5′-NT), ribonucleotide reductase catalytic and regulatory (RR1, RR2) subunits in bone marrow cells from 32 patients with Waldenström's Macroglobulinemia (WM) and small lymphocytic lymphoma (SLL) who received 2CdA-based chemotherapy. Responses to chemotherapy, were then correlated to the expression of these markers.ResultsAll 32 patients enrolled expressed lower levels of hCNT1 as compared to healthy donors. In univariate analysis, lower expression level of hCNT1 (p = 0.0021) and RR2 (p = 0.02) correlated with response to chemotherapy. In particular, patients with low levels of hCNT1 achieved inferior clinical response. No significant correlation between these genes expression and age, stage of disease was found. This study suggests that nucleotidase expression levels can be used to identify subgroups of WM and SLL patients who will likely respond differently to a 2CdA-based therapy.