Role of deoxycytidine kinase in the inhibitory activity of 5-substituted 2'-deoxycytidines and cytosine arabinosides on tumor cell growth

A number of 5-substituted derivatives of dCyd and 1-beta-D-arabinofuranosylcytosine (araC) have been evaluated for their inhibitory effects on the growth of three murine leukemia cell lines (L1210/0, L1210/BdUrd, and L1210/araC). The L1210/BdUrd and L1210/araC cell lines were selected from the parental L1210/0 cell line by their ability to grow at high concentrations of 5-bromo-2'-deoxyuridine and araC, respectively; the L1210/BdUrd cell line was deficient in dThd kinase activity, whereas the L1210/araC cell was deficient in dCyd kinase activity. The most effective inhibitors of L1210/0 cell proliferation were 5-fluoro-dCyd, araC, and 5-fluoro-araC. Their 50% inhibitory dose fell within the 0.001-0.015 micrograms/ml range. The 5-substituted araC analogues were much less inhibitory for L1210/araC cells but equally inhibitory for L1210/BdUrd as for the parental L1210/0 cell line. The role of dCyd kinase in the antitumor activity of the dCyd and araC analogues was further assessed by kinetic studies with dCyd kinase extracted from L1210/0 cells. All dCyd and araC analogues caused a competitive inhibition of dCyd kinase, the most potent inhibitor being 5-fluoro-dCyd (Ki/Km value 0.24). The Km of dCyd kinase from L1210/0 cells for dCyd was 23.1 microM as compared with 50 microM for araC. These values were increased to 53 and 182 microM, respectively, for the dCyd kinase isolated from L1210/araC cells.     

Current status of and future perspectives for platinum antitumor drugs

Cisplatin, a simple inorganic compound, has been one of the leading antitumor drugs for near 30 years. However, cisplatin has several drawbacks such as toxicity and drug resistance. Therefore, much attention has been focused on the development of new platinum complexes with improved pharmacological properties and a broader spectrum of activity to tumors. The recent advance of research on the molecular mechanisms of drug action and the cellular mechanisms of the emergence of resistance to cisplatin assists the rational design of new classes of platinum antitumor drugs, though details of both mechanisms still remain elusive. Information on DNA binding mode of platinum complexes, recognition and repair of DNA damage is instructive. Since several not cis isomers but trans isomers and not neutral complexes but cation complexes have been found active in vitro and in vivo, the early empirical structure-activity relationships of cisplatin analogues should be reevaluated. The hypothesis that platinum complexes which bind to DNA in a different manner will have different pharmacological properties has been tested, and now cationic multi-nuclear complexes and even trans-platinum complexes comprise unique classes of antitumor platinum-based agents with chemical and biological properties different from cisplatin. These new type platinum complexes are often effective to acquired cisplatin resistant tumor cells. In conclusion, the following complexes appear to offer great potential as new antitumor agents: (1) Complexes with distinctively different DNA interaction modes from cisplatin, which may circumvent intrinsic and acquired resistance to cisplatin through eluding the vigilance of DNA repair systems and (2) complexes with different tissue distribution or mechanisms of membrane transport which may exhibit a different spectrum of activity.     

Targets and effectors of the cellular response to aurora kinase inhibitor MK-0457 (VX-680) in imatinib sensitive and resistant chronic myelogenous leukemia

MK-0457 inhibits aurora, BCR-ABL and other kinases and may be clinically active in imatinib resistant leukemia. To define mediators of MK-0457 responsiveness, kinase inhibitory profiles were examined in multiple cell models of imatinib sensitive and resistant disease. Aurora and BCR-ABL kinase inhibition were consistently measured at 20-100 nM and 2-10 μM MK-0457, respectively, but expression of T315I-BCR-ABL and overexpression of Lyn kinase reduced MK-0457 sensitivity. Aurora kinase inhibition was associated with cell cycle restriction and p53 induction and p53-null cells were far less responsive to MK-0457, requiring BCR-ABL inhibitory concentrations for apoptotic activity. In wild-type p53 expressing CML cells MK-0457 sensitivity was modulation by alterations in p53 levels through HDM-2 inhibition and gene silencing. MK-0457 suppressed aurora kinase activity and induced apoptosis in imatinib resistant clinical specimens expressing T315I and other BCR-ABL mutations without effecting BCR-ABL kinase activity. Together, these results suggest that MK-0457 apoptotic activity in CML cells is primarily associated with aurora kinase inhibition but can be altered by multiple molecular changes associated with disease progression or acquisition of imatinib resistance.     

Selective activation of deoxycytidine kinase by thymidine-5'-thiosulphate and release by deoxycytidine in human lymphocytes

Deoxycytidine kinase (dCK) catalyses the rate-limiting step of the salvage of three natural deoxyribonucleosides as well as several therapeutic nucleoside analogues, which in turn can enhance its enzymatic activity [Biochem Pharmacol 56 (1998) 1175], improving the efficacy of the cytostatic therapy. Here, we measured the effect of the 5'-thiosulphate (5'-TS) derivatives of four deoxyribonucleosides (deoxyadenosine, deoxycytidine (dCyd), azidothymidine, thymidine) and two ribonucleosides (ribopurine, ribouridine (Urd)) on the activity of the two main salvage deoxynucleoside kinases, and on the salvage of dCyd and deoxythymidine (dThd). It turned out that only 2'-deoxythymidine-5'-thiosulphate (dThd-5'-TS) can potentiate the dCK activity, without influencing the thymidine kinase isoenzymes during short-time treatments of human peripheral blood and tonsillar lymphocytes. The enhancement of dCK activity by dThd-5'-TS can be reversed by dCyd, but dThd had no effect on the enzyme activation in cells. Neither dThd-5'-TS nor Urd-5'-TS had any effect on the dCK and thymidine kinase activities tested in cell-free extracts. The stimulation of dCK activity in cells was accompanied by an imbalance in the dThd and dCyd metabolism. The incorporation of 3H-dThd into DNA was suppressed by 90% in cells by dThd-5'-TS, while Urd-5'-TS only slightly influenced the same process. The 3H-dCyd incorporation into DNA was inhibited only to 50% of the control, while the 3H-dCyd labelling of the nucleotide fraction was enlarged in dThd-5'-TS-treated cells, as a consequence of the increased dCK activity. We suggest that the enhancement of dCK activity is a compensatory mechanism in cells that might be induced by different "inhibitors" of DNA synthesis leading to damage of DNA. The increased dCK activity is able to supply the repair of DNA with dNTPs in quiescent cells; this suggestion seems to be supported by the counteracting effect of extracellular dCyd, too.     

Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance

Mechanisms of acquired resistance to three purine analogues, 2-chloro-2'-deoxyadenosine (cladribine, CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (fludarabine, Fara-A), and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (clofarabine, CAFdA) were investigated in a human T-lymphoblastic leukemia cell line (CCRF-CEM). These analogues are pro-drugs and must be activated by deoxycytidine kinase (dCK). The CdA and CAFdA resistant cell lines exhibited increased resistance to the other nucleoside analogues activated by dCK. This was also the case for the Fara-A resistant cells, except that they were sensitive to CAFdA and guanosine analogues. The CdA and CAFdA resistant cells displayed a deficiency in dCK activity (to <5%) while the Fara-A resistant cells showed only a minor reduction of dCK activity (20% reduction). The activity of high K(m) 5'-nucleotidase (5'-NT) (cN-II) using IMP as substrate, was 2-fold elevated in the resistant cell lines. The amount of the small subunit R2 of ribonucleotide reductase (RR) was higher in the Fara-A resistant cells, which translated into a higher RR activity, while CdA and CAFdA cells had decreased activity compared to the parental cells. Expression of the recently identified RR subunit, p53R2 full-size protein, in CAFdA cells was low compared to parental cells, but a protein of lower molecular weight was detected in CdA and CAFdA cells. Co-incubation of Fara-A with the RR inhibitor 3,4-dihydroxybenzohydroxamic acid (didox) enhanced cytotoxicity in the Fara-A resistant cells by a factors of 20. Exposure of the cells to the nucleoside analogues studied here also caused structural and numerical instability of the chromosomes; the most profound changes were recorded for CAFdA cells, as demonstrated by SKY and CGH analysis. We conclude that down-regulation of dCK in cells resistant to CdA and CAFdA and increased activity of RR in cells resistant to Fara-A contribute to resistance.     

Substrate specificity of human deoxycytidine kinase toward antiviral 2',3'-dideoxynucleoside analogs.

Deoxycytidine (dCyd) kinase has been purified to homogeneity from human leukemic spleen, and the capacity of the enzyme to phosphorylate 2',3'-dideoxynucleoside (ddN) analogs that are clinically effective inhibitors of human immunodeficiency virus (HIV) replication was evaluated. Cytosine-containing ddN analogs, such as 2',3'-dideoxycytidine, 2',3'-dideoxy-2',3'-dehydrocytidine, and cytallene, were efficiently phosphorylated by dCyd kinase, while no phosphorylation of purine-containing ddN analogs was detected. dCyd kinase was completely inactive toward 2',3'-dideoxyadenosine (ddAdo), 2',3'-dideoxyinosine, 2',3'-dideoxyguanosine, and adenallene, although it was capable of phosphorylating both 2'-deoxyadenosine (dAdo) and 2'-deoxyguanosine (dGuo). The abilities of wild type and mutant human T lymphoblastoid CEM cells to accumulate ddAdo in situ and in vitro were also ascertained. Comparison of the abilities of intact wild type CEM cells and derivatives deficient in nucleoside transport, dCyd kinase, and/or adenosine (Ado) kinase to accumulate [3H]ddAdo-derived radioactivity revealed no significant differences among the wild type and mutant strains. However, ddAdo phosphorylating activity was decreased in extracts from Ado kinase-deficient cells but not in lysates prepared from cells genetically deficient in dCyd kinase activity. In comparative growth rate experiments, wild type, nucleoside transport-deficient, and dCyd kinase-deficient CEM cells were equally sensitive to ddAdo toxicity, while, interestingly, a deficiency in Ado kinase correlated with a 5-fold decreased growth sensitivity to the purine ddN. Insertion of an adenine phosphoribosyltransferase deficiency into the CEM cell lines did not influence ddAdo toxicity or incorporation rate. These results imply that Ado kinase may be an important factor in ddAdo phosphorylation by CEM cells. Furthermore, these studies demonstrate that cytosine- and purine-containing ddNs are transported and activated by independent pathways and, therefore, have important implications for anti-HIV therapy in that pyrimidine and purine ddNs might be used in combination for the treatment of acquired immunodeficiency syndrome.     

DNA interactions of new antitumor platinum complexes with trans geometry activated by a 2-metylbutylamine or sec-butylamine ligand

The global modification of mammalian and plasmid DNAs by novel platinum compounds, trans-[PtCl(2)(NH(3))(Am)], where Am=2 -methylbutylamine or sec-butylamine was investigated in cell-free media using various biochemical and biophysical methods. These modifications were analyzed in the context of the activity of these new compounds in several tumor cell lines including those resistant to antitumor cis-diamminedichloroplatinum(II) (cisplatin). The results showed that the replacement of one amine group by 2-methylbutylamine or sec-butylamine ligand in clinically ineffective trans-diamminedichloroplatinum(II) (transplatin) resulted in a radical enhancement of its activity in tumor cell lines so that they are more cytotoxic than cisplatin and exhibited significant antitumor activity including activity in cisplatin-resistant tumor cells. Importantly, this replacement also markedly altered DNA binding mode of transplatin and reduced the efficiency of repair systems to remove the adducts of the new analogues from DNA. The results support the view that one strategy to activate trans geometry in bifunctional platinum(II) compounds including circumvention of resistance to cisplatin may consist in a chemical modification of the ineffective transplatin which results in an increased efficiency to form DNA interstrand cross-links.     

Indolinones as promising scaffold as kinase inhibitors: a review

Kinases are probably the most important signaling enzymes, which represent about 20% of the druggable genome. Currently, more than 150 kinases are known. So, kinase inhibition therapy has become a very important area of drug research since most of our diseases are related to intra or intercellular signaling by kinases. Indole alkaloids are extensively studied for their biological activities in several pharmaceutical areas, including, for example, antitumor. Among this chemical family, indolinone displays very promising antitumor properties by inhibiting various kinase families. These small molecules have a low molecular weight and most of them bind to protein kinases competing with ATP for the ATP-binding site. This review focuses on the indolinone based drugs approved for the treatment of cancer, drugs under clinical trial and then chemical diversity of various synthetic analogues of indolinone and their metabolites as various kinase inhibitors. This review also focused on structural activity relationship (SAR), mechanisms of action and biological targets through which indolinone and its derivatives display their antitumor activity.     

Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance

Considering of novel biochemical modulation by some foods and beverages, we have performed screening for green tea components that have enhancing effects on doxorubicin (DOX) induced antitumor activity. Components, such as caffeine, theanine, (-)-epigallocatechin gallate (EGCG) and flavonoids have inhibitory effects on the DOX efflux from Ehrlich ascites carcinoma cells. Thus, it is suggested that EGCG and flavonoids may enhance DOX induced antitumor activity and increase the DOX concentrations in tumors through the inhibition of DOX efflux. It is expected that these components in green tea exhibit low toxicity and that there are few side effects of drinking green tea in combination with an antitumor agent. We think that the intake of a favorite beverage favors a positive mental attitude of a patient and increases the efficacy of the chemotherapeutic index, and that this efficacy is useful for improving the quality of life on cancer chemotherapy. In DOX resistant P388 leukemia cell bearing mice theanine increased the DOX induced efficacy through an increase in the DOX concentrations in the tumors. Theanine attacked the same transport process for DOX in both types of cells, elevated the DOX concentration and increased the DOX induced antitumor activity.     

Targeting DNA-PK overcomes acquired resistance to third-generation EGFR-TKI osimertinib in non-small-cell lung cancer

The third-generation of epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKIs),represented by osimertinib,has achieved remarkable clinical outcomes in the treatment of non-small-cell lung cancer(NSCLC)with EGFR mutation.However,resistance eventually emerges in most patients and the underlying molecular mechanisms remain to be fully understood.In this study,we generated an osimertinib-acquired resistant lung cancer model from a NSCLC cell line H1975 harboring EGFR L858R and T790M mutations.We found that the capacity of DNA damage repair was compromised in the osimertinib resistant cells,evidenced by increased levels ofγH2AX and higher intensity of the comet tail after withdrawal from cisplatin.Pharmacological inhibiting the activity or genetic knockdown the expression of DNA-PK,a key kinase in DNA damage response(DDR),sensitized the resistant cells to osimertinib.Combination of osimertinib with the DNA-PK inhibitor,PI-103,or NU7441,synergistically suppressed the proliferation of the resistant cells.Mechanistically,we revealed that DNA-PK inhibitor in combination with osimertinib resulted in prolonged DNA damage and cell cycle arrest.These findings shed new light on the mechanisms of osimertinib resistance in the aspect of DNA repair,and provide a rationale for targeting DNA-PK as a therapeutic strategy to overcome osimertinib-acquired resistance in NSCLC.     

Biochemical mechanisms of cisplatin cytotoxicity

Since the discovery by Rosenberg and collaborators of the antitumor activity of cisplatin 35 years ago, three platinum antitumor drugs (cisplatin, carboplatin and oxaliplatin) have enjoyed a huge clinical and commercial hit. Ever since the initial discovery of the anticancer activity of cisplatin, major efforts have been devoted to elucidate the biochemical mechanisms of antitumor activity of cisplatin in order to be able to rationally design novel platinum based drugs with superior pharmacological profiles. In this report we attempt to provide a current picture of the known facts pertaining to the mechanism of action of the drug, including those involved in drug uptake, DNA damage signals transduction, and cell death through apoptosis or necrosis. A deep knowledge of the biochemical mechanisms, which are triggered in the tumor cell in response to cisplatin injury not only may lead to the design of more efficient platinum antitumor drugs but also may provide new therapeutic strategies based on the biochemical modulation of cisplatin activity.     

Differential modulation of 1-beta-D-arabinofuranosylcytosine metabolism by hydroxyurea in human leukemic cell lines

The ability of hydroxyurea (HU) to modulate 1-beta-D-arabinofuranosylcytosine (Ara-C) metabolism was investigated in human leukemic cell lines. Exposure of HL-60 cells to 1 mM HU enhanced the accumulation of Ara-CTP up to 2.5-fold, whereas HU did not have significant effects on Ara-C metabolism in CEM cells. In addition, two adenine nucleosides, deoxyadenosine (dAdo) and 9-beta-D-arabinofuranosyladenine (Ara-A), which are known to be activated by deoxycytidine (dCyd) kinase as Ara-C, were more effectively phosphorylated after the addition of HU only in HL-60 cells. However, the changes of intracellular dCTP and TTP pools induced by HU, i.e. decrease in dCTP and increase in TTP, were the same in both cell lines. Finally, dCyd production under normal culture conditions was at least 3- to 4-fold higher in HL-60 cells and was inhibited significantly by HU administration. These results suggest that the modulation of Ara-C metabolism by HU occurs at the level of dCyd kinase through the regulation of de novo dCyd generation.     

Modifications of DNA by platinum complexes. Relation to resistance of tumors to platinum antitumor drugs.

The importance of platinum drugs in cancer chemotherapy is underscored by the clinical success of cisplatin [cis-diamminedichloroplatinum(II)] and its analogues and by clinical trials of other, less toxic platinum complexes that are active against resistant tumors. The antitumor effect of platinum complexes is believed to result from their ability to form various types of adducts with DNA. Nevertheless, drug resistance can occur by several ways: increased drug efflux, drug inactivation, alterations in drug target, processing of drug-induced damage, and evasion of apoptosis. This review focuses on mechanisms of resistance and sensitivity of tumors to conventional cisplatin associated with DNA modifications. We also discuss molecular mechanisms underlying resistance and sensitivity of tumors to the new platinum compounds synthesized with the goal to overcome resistance of tumors to established platinum drugs. Importantly, a number of new platinum compounds were designed to test the hypothesis that there is a correlation between the extent of resistance of tumors to these agents and their ability to induce a certain kind of damage or conformational change in DNA. Hence, information on DNA-binding modes, as well as recognition and repair of DNA damage is discussed, since this information may be exploited for improved structure-activity relationships.     

Neurotensin attenuates dopamine D2 agonist quinpirole-induced inhibition of midbrain dopamine neurons

The effects of intracerebroventricular (i.c.v.) administration of NT on the activity of midbrain DA neurons were studied in rats using single unit recording techniques. NT (i.c.v., 50 micrograms) was found to have no significant effect on the spontaneous activity of DA cells. On the other hand, NT treatment significantly attenuated the inhibitory effect of quinpirole (i.v.), a specific D2 agonist, on a subpopulation of DA cells. This result is consistent with previous behavioral and biochemical studies suggesting that NT may produce some of its effects through modulation of central DA systems.     

Point mutations in human guanylate kinase account for acquired resistance to anticancer nucleotide analogue PMEG

Acyclic nucleotide analogue PMEG represents promising drug candidate against lymphomas. In the present work we describe the ability of PMEG to induce resistance and we elucidate the mechanisms involved in this process. CCRF-CEM T-lymphoblastic cells resistant to either PMEG or its 6-amino congener PMEDAP were prepared and assayed for the expression of membrane transporters, PMEG and PMEDAP uptake and intracellular metabolism. Genes for guanylate kinase (GUK) and adenylate kinase (AK) isolated from PMEG- and PMEDAP-resistant cells were sequenced and cloned into mammalian expression vectors. PMEG-resistant cells were transfected with GUK vectors and catalytic activities of GUKs isolated from PMEG-sensitive and resistant cells were compared. PMEG phosphorylation to PMEG mono- and diphosphate was completely impaired in resistant cells. GUK obtained from PMEG-resistant cells revealed two point mutations S³⁵N V¹⁶⁸F that significantly suppressed its catalytic activity. Transfection of resistant cells with wtGUK led to the recovery of phosphorylating activity as well as sensitivity towards PMEG cytotoxicity. No differences in PMEG uptake have been found between sensitive and resistant cells. In contrast to GUK no changes in primary sequence of AK isolated from PMEDAP resistant cells were identified. Therefore, resistance induced by PMEDAP appears to be conferred by other mechanisms. In conclusion, we have identified GUK as the sole molecular target for the development of acquired resistance to the cytotoxic nucleotide PMEG. Therefore, PMEG is unlikely to cause cross-resistance in combination therapeutic protocols with most other commonly used anticancer drugs.     

A crucial role of uridine/cytidine kinase 2 in antitumor activity of 3'-ethynyl nucleosides.

The antitumor 3'-ethynyl nucleosides, 1-(3-C-ethynyl-beta-D-ribopentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-beta-D-ribopentofuranosyl)uridine (EUrd), are potent inhibitors of RNA polymerases and show excellent antitumor activity against various human solid tumors in xenograft models. ECyd is being investigated in phase I clinical trials as a novel anticancer drug possessing a unique antitumor action. ECyd and EUrd require the activity of uridine/cytidine kinase (UCK) to produce the corresponding active metabolite. The UCK family consists of two members, UCK1 and UCK2, and both UCKs are expressed in many tumor cells. It was unclear, however, whether UCK1 or UCK2 is responsible for the phosphorylation of the 3'-ethynyl nucleosides. We therefore established cell lines that are highly resistant to the 3'-ethynyl nucleosides from human fibrosarcoma HT-1080 and gastric carcinoma NUGC-3. All the resistant cell lines showed a high cross-resistance to ECyd and EUrd. As a result of cDNA sequence analysis, we found that UCK2 mRNA expressed in EUrd-resistant HT-1080 cells has a 98-base pair deletion of exon 5, whereas EUrd-resistant NUGC-3 cells were harboring the point mutation at nucleotide position 484 (C to T) within exon 4 of UCK2 mRNA. This mutation was confirmed by genome sequence analysis of the UCK2 gene. Moreover, the expression of UCK2 protein was decreased in these resistant cells. In contrast, no mutation in the mRNA or differences in protein expression levels of UCK1 were shown in the EUrd-resistant HT-1080 and NUGC-3 cells. These results suggest that UCK2 is responsible for the phosphorylation and activation of the antitumor 3'-ethynyl nucleosides.     

Synthesis and biological activity of analogues of the antimicrotubule agent N,beta,beta-trimethyl-L-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]- N(1),3-dimethyl-L-valinamide (HTI-286)

Hemiasterlin, a tripeptide isolated from marine sponges, induces microtubule depolymerization and mitotic arrest in cells. HTI-286, an analogue from an initial study of the hemiasterlins, is presently in clinical trials. In addition to its potent antitumor effects, 2 has the advantage of circumventing the P-glycoprotein-mediated resistance that hampers the efficacy of other antimicrotubule agents such as paclitaxel and vincristine in animal models. This paper describes an in-depth study of the structure--activity relationships of analogues of 2, their effects on microtubule polymerization, and their in vitro and in vivo anticancer activity. Regions of the molecule necessary for potent activity are identified. Groups tolerant of modification, leading to novel analogues, are reported. Potent analogues identified through in vivo studies in tumor xenograft models include one superior analogue, HTI-042.     

Characterization of a human carcinoma cell line selected for resistance to the farnesyl transferase inhibitor 4-(2-(4-(8-chloro-3,10-dibromo-6,11-dihydro-5H-benzo-(5,6)-cyclohepta(1,2-b)-pyridin-11(R)-yl)-1-piperidinyl)-2-oxo-ethyl)-1-piperidinecarboxamide (SCH66336)

Farnesyl protein transferase inhibitors (FTIs) have demonstrated clinical activity in certain solid tumors and hematological malignancies. Little is known about mechanisms of resistance to these agents. To provide a basis for better understanding FTI resistance, the colorectal carcinoma cell line HCT 116 was selected by stepwise exposure to increasing 4-(2-(4-(8-chloro-3,10-dibromo-6,11-dihydro-5H-benzo-(5,6)-cyclohepta(1,2-b)-pyridin-11(R)-yl)-1-piperidinyl)-2-oxo-ethyl)-1-piperidinecarboxamide (SCH66336) concentrations. The resulting line, HCT 116R, was 100-fold resistant to SCH66336 and other FTIs, including methyl {N-[2-phenyl-4-N[2(R)-amino-3-mercaptopropylamino] benzoyl]}-methionate (FTI-277), but was less than 2-fold resistant to the standard agents gemcitabine, cisplatin, and paclitaxel. Accumulation of the unfarnesylated forms of prelamin A and HDJ-2, two substrates that reflect farnesyl transferase inhibition, was similar in FTI-treated parental and HCT 116R cells, indicating that alterations in drug uptake or inhibition of farnesyl protein transferase is not the mechanism of resistance. Changes in signal-transduction pathways that might account for this resistance were examined by immunoblotting and confirmed pharmacologically. There was no difference in activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase pathway or sensitivity to the MEK1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059) in HCT 116R cells. In contrast, increased phosphorylation of the molecular target of rapamycin (mTOR) and its downstream target p70 S6 kinase and increased levels of Akt1 and Akt2 were demonstrated in HCT 116R cells. Further experiments demonstrated that the mTOR inhibitor rapamycin selectively sensitized HCT 116R cells to SCH66336 but not to gemcitabine, cisplatin, or paclitaxel. These findings provide evidence that alterations in the phosphatidylinositol-3 kinase/Akt pathway can contribute to FTI resistance and suggest a potential strategy for overcoming this resistance.