Down–Regulation of Human Deoxyuridine Triphosphate Nucleotidohydrolase (dUTPase) Using Small Interfering RNA (siRNA)

A small interfering double stranded RNA molecule (siRNA, 21 bp) corresponding to a portion (nucleotides 337 to 357) of domain 3 of the human dUTPase was synthesized and used to determine whether it could down‐regulate dUTPase activity in human cells. Transfection of the siRNA into HeLa and HT29 cells resulted in a 56 ± 3.6% decrease in dUTPase activity, while transfection of SW620 cells resulted in a 27 ± 6% decrease in dUTPase activity when compared to non‐treated controls.     

Inhibition of MMP-2 gene expression with small interfering RNA in rabbit vascular smooth muscle cells

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs). Using small interfering RNA (siRNA), we evaluated the effect of MMP-2 inhibition in VSMCs in vitro and ex vivo. Rabbit VSMCs were transfected in vitro with 50 nmol/l MMP-2 siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in approximately 80% of VSMCs. MMP-2 mRNA levels evaluated by real-time RT-PCR, pro-MMP-2 activity from conditioned culture media evaluated by gelatin zymography, and VSMC migration were reduced by 44 +/- 19%, 43 +/- 14%, and 36 +/- 14%, respectively, in MMP-2 siRNA-transfected compared with scramble siRNA-transfected VSMCs (P < 0.005 for all). Ex vivo MMP-2 siRNA transfection was performed 2 wk after balloon injury of hypercholesterolemic rabbit carotid arteries. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture medium demonstrated a significant decrease of pro-MMP-2 activity in MMP-2 siRNA-transfected compared with scramble siRNA-transfected arteries (P < 0.01). Overall, our results demonstrate that in vitro MMP-2 siRNA transfection in VSMCs markedly inhibits MMP-2 gene expression and VSMC migration and that ex vivo delivery of MMP-2 siRNA in balloon-injured arteries reduces pro-MMP-2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.     

Critical role for c-FLIP(L) on Fas resistance in colon carcinoma cell line HT-29

The cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein-long form (c-FLIP(L)) is a key regulator of Fas signaling, although owing a dominant-negative homologue of caspase-8, the role of c-FLIP(L) remains controversial. In the present study, two pairs of small interfering RNA (siRNA) directed against c-FLIP(L) were used to assess the effect of c-FLIP(L) on Fas-mediated apoptosis of colon carcinoma in vitro. HT-29 cell line was selected for overexpression of c-FLIP(L) and Fas with RT-PCR and flow cytometry analyses. After electroporation, the mRNA level of c-FLIP(L) was significantly decreased (control siRNA versus c-FLIP(L) siRNA, 77.97+/-5.61% versus 26.22+/-3.79%) and the maximum interfering efficiency was around 66.49% using semi-quantitative RT-PCR analysis. Knockdown of c-FLIP(L) with the specific siRNA sensitized colon carcinoma cells to Fas-mediated apoptosis (control siRNA versus c-FLIP(L) siRNA, 5.68+/-2.11% versus 29.50+/-2.27%) using DNA content analysis and Annexin V-FITC analysis. In conclusion, our study indicated that c-FLIP(L) might be a suppressor of Fas-mediated apoptosis in Fas antigen expressing colon carcinoma and therefore a potential target for novel anticancer therapies.     

Mitogen induction of deoxyuridine triphosphatase activity in human T and B lymphocytes

Deoxyuridine triphosphatase (dUTPase; deoxyuridine diphosphohydrolase; EC 3.6.1.23) activity during mitogen stimulation was investigated in human T-cell and B-cell enriched mononuclear leucocyte fractions as well as in a mixed lymphocyte population. The dUTPase activity was very low in the resting peripheral blood lymphocytes. A remarkable enhancement of enzymatic activity was observed when cells were stimulated with different mitogens; T-cells and non-separated lymphocytes with phytohaemagglutinin, and the B-cell enriched fraction with pokeweed mitogen. There was a positive correlation between dUTPase activity and the enhancement of macromolecule synthesis (protein and RNA). In particular, a highly significant correlation was observed between dUTPase activity and DNA synthesis in the three human lymphocyte populations studied. This supports the view that the enzyme dUTPase may have a significant role in cellular proliferation. The physiological role of the enzyme is discussed.     

Knockdown of cytosolic 5'-nucleotidase II (cN-II) reveals that its activity is essential for survival in astrocytoma cells

IMP preferring cytosolic 5'-nucleotidase (cN-II) is an ubiquitous nucleotide hydrolysing enzyme. The enzyme is widely distributed and its amino acid sequence is highly conserved among vertebrates. Fluctuations of cN-II activity have been associated with the pathogenesis of neurological disorders. The enzyme appears to be involved in the regulation of the intracellular availability of the purine precursor IMP and also of GMP and AMP, but the contribution of this activity and of its regulation to cell metabolism and to CNS cell functions remains uncertain. To address this issue, we used a vector based short hairpin RNA (shRNA) strategy to knockdown cN-II activity in human astrocytoma cells. Our results demonstrated that 53 h after transduction, cN-II mRNA was reduced to 17.9+/-0.03% of control cells. 19 h later enzyme activity was decreased from 0.7+/-0.026 mU/mg in control ADF cells to 0.45+/-0.046 mU/mg, while cell viability (evaluated by the MTT reduction assay) decreased up to 0.59+/-0.01 (fold vs control) and caspase 3 activity increased from 136+/-5.8 pmol min(-1) mg(-1) in control cells to 639+/-37.5 pmol min(-1) mg(-1) in silenced cells, thus demonstrating that cN-II is essential for cell survival. The decrease of enzyme activity causes apoptosis of the cultured cells without altering intracellular nucleotide and nucleoside concentration or energy charge. Since cN-II is highly expressed in tumour cells, our finding offers a new possible therapeutical approach especially against primary brain tumours such as glioblastoma, and to ameliorate chemotherapy against leukemia.     

Human catecholamine sulfotransferase (SULT1A3) pharmacogenetics: functional genetic polymorphism

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5'-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 +/- 4.5% (mean +/- SEM) of the enzyme activity of the wild-type (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 +/- 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.     

Natural killer (NK)-resistant human lung cancer cells are lysed by recombinant interleukin-2-activated NK cells

Natural killer (NK) cells are active in host defence against tumors. In order to determine if NK cells have the capacity to lyse human lung cancer cells, we evaluated blood NK cell activity against human lung carcinoma lines representing each of the commonest histological types of lung cancer, NCI-H157 (large cell), LICM107 and NCI-H146 (small cell), NCI-H226 (squamous cell), and LICM26 (adeno), and compared the results to their activity against a standard NK-sensitive target, K562, using a 16-hr 51Cr-release assay. At effector to target (E:T) ratios up to 50:1, NK activity was very low against each of the lung cancer cell lines compared to the K562 cells (NCI-H157 10 +/- 2%, LICM107 12 +/- 2%, NCI-H146 14 +/- 5%, NCI-H226 8 +/- 5%, and LICM26 7 +/- 3%, compared to K562 60 +/- 3%, P less than 0.001, for each compared to K562 cells). Recombinant interleukin 2 (IL-2) produced a dose-dependent augmentation of NK activity against each of the lung cancer cell lines, with doses as low as 0.25 U/ml being effective. The highest level of boosting was seen against NCI-H157 cells where NK activity (E:T, 50:1, IL-2, 250 U/ml) increased from 9 +/- 2 to 56 +/- 7%, P less than 0.001). Only brief exposure to IL-2 was necessary for augmentation to occur, with as little as 5 min being required for activation, although increased exposure times produced increased levels of augmentation. NK cells appeared to be the IL-2-responsive lytic cell population in these experiments as Leu 11b-depleted lymphocytes expressed little IL-2-mediated augmentation of activity against these target cells, and most of this IL-2-mediated augmentation of activity was located in the large granular lymphocyte-enriched fraction of the lymphocyte population. We conclude that normal blood NK cell activity against human lung cancer cell lines is low but that this activity can be markedly augmented by brief exposure of NK cells to low doses of recombinant IL-2, suggesting a potential role for IL-2 in the immunotherapy of human lung cancer.     

Functional proteomics of resveratrol-induced colon cancer cell apoptosis: caspase-6-mediated cleavage of lamin A is a major signaling loop

The study investigated the molecular basis of resveratrol (RSV)-evoked apoptosis in four (Bax+/-, Bax-/-, p53+/+, and p53-/-) HCT116 colon cancer cell lines. RSV induced apoptosis in all the cells in a dose-dependent manner; however, Bax+/- and p53+/+ cells were more susceptible than their knockout counterparts (Bax-/- and p53-/-, respectively). Using Bax+/- cells as a model, proteomic analysis revealed four RSV-responsive events: fragmentation of lamin A/C protein; increase in concentration of a more basic isoelectric variant of the ribosomal protein P0; and decrease in concentration of dUTPase as well as stathmin 1. Lamin A cleavage in response to RSV treatment was confirmed using Western blot analysis. Caspase-6 was activated, which was evidenced by cleavage and accumulation in active form of caspase-6 as well as upregulation of the protease activity. RSV-elicited lamin A cleavage and apoptosis were entirely abrogated by the peptide inhibitors of caspase-6. Likewise, partial knockdown of caspase-6 expression using small interfering RNA resulted in significant inhibition of RSV-elicited lamin A cleavage and apoptosis. Furthermore, the lower apoptosis sensitivity of the knockout cells (Bax-/- and p53-/-) correlated with the relatively reduced processing of caspase-6 and lamin A cleavage. Taken together, these data highlight the critical role of caspase-6 and its cleavage of lamin A in apoptotic signaling triggered by RSV in the colon carcinoma cells, which can be activated in the absence of Bax or p53.     

PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1)

Although the phosphorylation-dependent activation of the Na-K-Cl cotransporter (NKCC1) has been previously well documented, the identity of the kinase(s) responsible for this regulation has proven elusive. Recently, Piechotta et al. (Piechotta, K., Lu, J., and Delpire, E. (2002) J. Biol. Chem. 277, 50812-50819) reported the binding of PASK (also referred as SPAK (STE20/SPS1-related proline-alanine-rich kinase)) and OSR1 (oxidative stress response kinase) to cation-chloride cotransporters KCC3, NKCC1, and NKCC2. In this report, we show that overexpression of a kinase inactive, dominant negative (DN) PASK mutant drastically reduces both shark (60 +/- 5%) and human (80 +/- 3%) NKCC1 activation. Overexpression of wild type PASK causes a small (sNKCC1 22 +/- 8% p < 0.05, hNKCC1 12 +/- 3% p < 0.01) but significant increase in shark and human cotransporter activity in HEK cells. Importantly, DNPASK also inhibits the phosphorylation of two threonines, contained in the previously described N-terminal regulatory domain. We additionally show the near complete restoration of NKCC1 activity in the presence of the protein phosphatase type 1 inhibitor calyculin A, demonstrating that DNPASK inhibition results from an alteration in kinase/phosphatase dynamics rather than from a decrease in functional cotransporter expression. Coimmunoprecipitation assays confirm PASK binding to NKCC1 in transfected HEK cells and further suggest that this binding is not a regulated event; neither PASK nor NKCC1 activity affects the association. In cells preloaded with 32Pi, the phosphorylation of PASK, but not DNPASK, coincides with that of NKCC1 and increases 5.5 +/- 0.36-fold in low [Cl]e. These data conclusively link PASK with the phosphorylation and activation of NKCC1.     

Purification and properties of the deoxyuridine triphosphate nucleotidohydrolase enzyme derived from HeLa S3 cells. Comparison to a distinct dUTP nucleotidohydrolase induced in herpes simplex virus-infected HeLa S3 cells

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) (EC 3.6.1.23) derived from HeLa S3 cells has been purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of about 16,000 nmol of dUMP hydrolyzed per min/mg of protein. The dUTPase enzyme derived from HeLa S3 cells appears to be composed to two equal molecular mass subunits, each being about 22,500 daltons. Association of these subunits to produce a 45,000-dalton protein is promoted by MgCl2. In the presence of EDTA enzyme activity is abolished and the enzyme dissociates into its monomeric form. MgCl2 will completely reverse the inhibition caused by EDTA and promote subunit association. MnCl2 will also promote association of the dUTPase subunits. However, MnCl2 will not completely reverse inhibition by EDTA. In addition, purified dUTPase, extensively dialyzed to remove contaminating ions, is activated almost 2-fold by the addition of 5 mM MgCl2. In contrast, addition of 5 mM MnCl2 to the dialyzed enzyme preparation will cause more than a 50% decrease in enzyme activity. This data indicates that Mg2+ is the natural prosthetic group for this enzyme. The Km value of dUTP for the purified enzyme is 3 X 10(-6) M in the presence of MgCl2. The turnover number for this enzyme has been calculated to be 550 molecules of dUTP hydrolyzed per min under standard assay conditions. Infection of HeLa S3 cells with herpes simplex type 1 virus induces a distinct species of dUTPase. This new species of dUTPase has an isoelectric point of 8.0, compared to an isoelectric point in the range of 5.7 to 6.5 for the HeLa S3 dUTPase. Molecular weight determinations of this new species of dUTPase indicate that the native enzyme is monomeric with a molecular weight of about 35,000. The virally induced dUTPase is inhibited by EDTA and this inhibition is reversed by MgCl2. Unlike the HeLa S3 dUTPase, the virally induced enzyme does not appear to be composed of subunits.     

Lysosomal enzymes and natural killer activity

The natural killer activity (NKA) of human mononuclear cells and the activity of the lysosomal enzymes of these cells (arylsulfatase, acid phosphatase and beta-glucuronidase) has been studied in norm and under human lung cancer. The mononuclear cells were isolated from peripheral blood of 10 healthy donors and 20 patients with lung cancer of II-III stages. Under the action of mononuclear cells on the target cells (human erythroleukosis cells K-562 labeled with 3H-uridine) the NKA of mononuclear cells of patients was seen to decrease (cytotoxic index = 54.8 +/- 6.4%), in comparison with that of healthy donors (cytotoxic index = 65.1 +/- 4.5%). Simultaneously a decrease in arylsulfatase activity (0.05 +/- 0.01 nmoles/10(6) cells/min) was found in comparison with the control value (0.11 +/- 0.01 nmoles/10(6) cells/min). In 2-3 weeks after the operation the NKA value (cytotoxic index = 50.2 +/- 5.8%) was restored and arylsulfatase activity (0.09 +/- 0.02 nmoles/10(6) cells/min) was increased. There was no correlation between the NKA value and the activities of acid phosphatase and beta-glucuronidase. The parallelism observed between changes in NKA value and arylsulfatase activity may suggest a possible participation of this enzyme in the killing mechanism at the stage of cerebroside sulfate ester degradation of the target cell membrane to initiate the lytic events.     

Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis

RNA interference (RNAi) treatment of monkey COS-7 cells, a cell line that lacks nonmuscle myosin heavy chain II-A (NMHC II-A) but contains NMHC II-B and II-C, was used to investigate the participation of NMHC isoforms in cytokinesis. We specifically suppressed the expression of NMHC II-B or II-C using 21 nucleotide small interfering RNA (siRNA) duplexes. Down-regulation of NMHC II-B protein expression to 10.2 +/- 0.7% inhibited COS-7 cell proliferation by 50% in the RNAi-treated cells compared with control cells. Moreover, whereas 8.7 +/- 1.0% of control cells were multinucleated, 62.4 +/- 8.8% of the NMHC II-B RNAi-treated cells were multinucleated 72 h after transfection. The RNAi-treated cells had increased surface areas and, unlike control cells, lacked actin stress fibers. Treatment of the COS-7 cells with NMHC II-C siRNA decreased NMHC II-C expression to 5.2 +/- 0.1% compared with the endogenous content of II-C; however, down-regulation of NMHC II-C did not cause increased multinucleation. Immunoblot analysis using a pan-myosin antibody showed that the content of NMHC II-C was less than one-twentieth the amount of NMHC II-B, thereby explaining the lack of response to II-C siRNA. Introducing green fluorescent protein (GFP)-tagged NMHC II isoforms into II-B siRNA-treated cells resulted in reduction of multinucleation from 62.4 +/- 8.8% to 17.8 +/- 2.2% using GFP-NMHC II-B, to 29.8 +/- 7.4% using GFP-NMHC II-A, and to 34.1 +/- 8.6% using NMHC II-C-GFP. These studies have shown that expression of endogenous NMHC II-C in COS-7 cells is insufficient for normal cytokinesis and that exogenous NMHC II-A and NMHC II-C can, at least partially, rescue the defect in cytokinesis due to the loss of NMHC II-B.     

Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca(2+)-independent phospholipase A(2).

Platelet activating factor (PAF) is a potent lipid autocoid that is rapidly synthesized and presented on the surface of endothelial cells following thrombin stimulation. PAF production may occur via de novo synthesis or by the combined direct action of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase or via the remodeling pathway. This study was undertaken to define the role of PLA(2) and plasmalogen phospholipid hydrolysis in PAF synthesis in thrombin-treated human umbilical artery endothelial cells (HUAEC). Basal PLA(2) activity in HUAEC was primarily found to be Ca(2+)-independent (iPLA(2)), membrane-associated, and selective for arachidonylated plasmenylcholine substrate. Thrombin stimulation of HUAEC resulted in a preferential 3-fold increase in membrane-associated iPLA(2) activity utilizing plasmenylcholine substrates with a minimal increase in activity with alkylacyl glycerophospholipids. No change in cystolic iPLA(2) activity in thrombin-stimulated HUAEC was observed. The thrombin-stimulated activation of iPLA(2) and associated hydrolysis of plasmalogen phospholipids was accompanied by increased levels of arachidonic acid (from 1.1 +/- 0.1 to 2.8 +/- 0.1%) and prostacyclin release (from 38 +/- 12 to 512 +/- 24%) as well as an increased level of production of lysoplasmenylcholine (from 0.6 +/- 0.1 to 2.1 +/- 0.3 nmol/mg of protein), lysophosphatidylcholine (from 0.3 +/- 0.1 to 0.6 +/- 0.1 nmol/mg of protein), and PAF (from 790 +/- 108 to 3380 +/- 306 dpm). Inhibition of iPLA(2) with bromoenol lactone resulted in inhibition of iPLA(2) activity, plasmalogen phospholipid hydrolysis, production of choline lysophospholipids, and PAF synthesis. These data indicate that PAF production requires iPLA(2) activation in thrombin-stimulated HUAEC and may occur through the CoA-independent transacylase remodeling pathway rather than as a direct result of the PLA(2)-catalyzed hydrolysis of membrane alkylacyl glycerophosphocholine.     

Enhanced sensitivity of human hepatoma cells to 5-fluorouracil by small interfering RNA targeting Bcl-2

This study was designed to reveal whether the apoptosis induced in human hepatocellular carcinoma (HCC) cell lines by 5-fluorouracil (5-FU) could be enhanced by transfecting Bcl-2 small interfering RNA (siRNA). Bcl-2 siRNA and control siRNA were transfected into cells following treatment with or without 5-FU. Suppression of Bcl-2 expression was confirmed by Western blotting; cell viability was evaluated by MTS assay, and the occurrence of apoptosis in cells was evaluated by apoptosis assay. Expression of Bcl-2 protein after transfection of 20 nM Bcl-2 siRNA was significantly lower than that of control. Incubation of all cell lines with Bcl-2 siRNA reduced cell viability 96 h after 5-FU treatment compared with all other controls: Huh-7 (P < 0.01), Huh-7 with hepatitis C replicon (P < 0.01), HepG2 (P < 0.01), HLE (P < 0.05). Moreover, the proportion of apoptosis in control siRNA, Bcl-2 siRNA, control siRNA prior to 5-FU treatment, and Bcl-2 siRNA prior to 5-FU treatment groups were (4.6 +/- 2.3)%, (7.5 +/- 0.5)%, (6.0 +/- 2.1)%, and (19.5 +/- 0.86)%, respectively. The Bcl-2 siRNA prior to 5-FU treatment group showed the strongest effect of inducing apoptosis. In conclusion, the combination Bcl-2 siRNA and 5-FU might represent a new therapeutic option for HCC.     

Presence of dUTPase in the Various Human Endogenous Retrovirus K (HERV-K) Families

Various retroviruses have been shown to encode dUTPase. The overall phylogeny of dUTPase is unclear, though. The human genome contains a significant amount of human endogenous retroviruses (HERV) representing fossilized sequences of ancient exogenous retroviruses. A few HERV families have been reported to harbor dUTPase domains. We surveyed the various HERV families for the presence of dUTPase and found that ancestors of all HERV-K families but one encoded dUTPase. With two exceptions phylogenetic analysis shows a monophyletic origin of dUTPase for the different HERV-K dUTPases. Sequences of consensus dUTPase domains suggest that the various exogenous ancestors of HERV-K once encoded active enzymes. Our analysis provides informations on dUTPase phylogeny and further shows that endogenous retroviruses provide important informations regarding retrovirus evolution.     

Mutations in accessory DNA replicating functions alter the relative mutation frequency of herpes simplex virus type 1 strains in cultured murine cells.

The contribution of the herpes simplex virus type 1 (HSV-1)-encoded uracil DNA glycosylase (UNG), thymidine kinase (TK), and dUTPase to the relative mutant frequency (RMF) of the virus in cultured murine cells was examined. A panel of HSV-1 mutants that lacked singly or doubly the UNG, TK, or dUTPase activity were generated by disruption of the enzyme coding regions with the Escherichia coli beta-galactosidase (beta-gal) gene in strain 17syn+. To establish a baseline RMF of strain 17syn+, the beta-gal gene was inserted into the UL3 locus. In all of the viruses, the beta-gal insert served as a phenotypic marker of RMF. A mutant plaque was identified by the lack of beta-gal activity and, in selected cases, positive in situ hybridization for beta-gal sequences. Replication kinetics in NIH 3T3 cells demonstrated that all of the mutants replicated efficiently, generating stocks with equivalent titers. Two independently generated UL3-beta-gal viruses were examined and established a baseline RMF of approximately 0.5% in both NIH 3T3 and LM TK- cells. Loss of dUTPase activity resulted in viruses with fivefold-increased RMFs, indicating that the HSV-1 dUTPase has an antimutator function. The RMF observed for the tk- viruses was reduced as much as 40-fold (RMF of 0.02%), suggesting that the viral TK is a mutator activity. The RMF of two independent UNG- viruses showed no significant difference from the baseline RMF in limited passage; however, following successive passage, the data suggested that UNG activity serves as an antimutator. These results have implications for the natural history of HSV and the development of antiviral therapies.     

Determinants of differential doxorubicin sensitivity between SCLC and NSCLC

Doxorubicin (DOX) transport activity of Ral-interacting protein (RLIP76) in non-small cell lung cancer (NSCLC) is approximately twice that of in small cell lung cancer (SCLC). Since protein-kinase-C (PKC)alpha mediated phosphorylation of RLIP76 causes doubling of the specific activity of RLIP76, and NSCLC cells are known to have greater PKCalpha activity, we examined the contribution of PKC mediated phosphorylation of RLIP76 towards intrinsic DOX-resistance in human NSCLC. Expression of a deletion mutant RLIP76(delPKCalpha-sites) followed by depletion of the wild-type RLIP76 using a siRNA targeted at one of the deleted regions resulted in generation of cells expressing only the mutant protein, which could not be phosphorylated by PKCalpha. DOX-transport activity of the mutant RLIP76 purified from NSCLC and SCLC was similar and comparable to that of RLIP76 purified from the wild-type SCLC. However, this activity was significantly lower than that of RLIP76 purified from the wild-type NSCLC. After siRNA mediated depletion of PKCalpha, DOX-transport activities of RLIP76 purified from SCLC and NSCLC were indistinguishable. Depletion of PKCalpha inhibited the growth of NSCLC more than SCLC cells (70+/-3% vs. 43+/-5%, respectively). PKCalpha-depletion lowered the IC(50) of NSCLC cell lines for DOX to the same level as that observed for SCLC. RLIP76(-/-) mouse embryonic fibroblasts (MEFs) were significantly more sensitive to DOX as compared with RLIP76(+/+) MEFs (IC(50) 25 vs. 125nM, respectively). However, PKCalpha-depletion did not affect DOX-cytotoxicity towards RLIP76(-/-) MEFs, as opposed to RLIP76(+/+) MEFs which were sensitized by 2.2-fold. These results demonstrate that RLIP76 is a primary determinant of DOX-resistance, and that PKCalpha mediated accumulation defect and DOX-resistance in NSCLC is primarily due to differential phosphorylation of RLIP76 in SCLC and NSCLC.