Inhibition of activation of dsRNA-dependent protein kinase and tumour growth inhibition

Inhibition of dsRNA-activated protein kinase (PKR), not only attenuates muscle atrophy in a murine model of cancer cachexia (MAC16), but it also inhibits tumour growth. In vitro the PKR inhibitor maximally inhibited growth of MAC16 tumour cells at a concentration of 200 nM, which was also maximally effective in attenuating phosphorylation of PKR and of eukaryotic initiation factor (eIF)2 on the a-subunit. There was no effect on the growth of the MAC13 tumour, which does not induce cachexia, even at concentrations up to 1,000 nM. There was constitutive phosphorylation of PKR and eIF2a in the MAC16, but not in the MAC13 tumour, while levels of total PKR and eIF2a were similar. There was constitutive upregulation of nuclear factor-?B (NF-?B) in the MAC16 tumour only, and this was attenuated by the PKR inhibitor, suggesting that it arose from activation of PKR. In MAC16 alone the PKR inhibitor also attenuated expression of the 20S proteasome. The PKR inhibitor potentiated the cytotoxicity of both 5-fluorouracil and gemcitabine to MAC16 cells in vitro. These results suggest that inhibitors of PKR may be useful therapeutic agents against tumours showing increased expression of PKR and constitutive activation of NF-?B, and may also prove useful in sensitising tumours to standard chemotherapeutic agents.

Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

Atrophy of skeletal muscle is due to a depression in protein synthesis and an increase in degradation. Studies in vitro have suggested that activation of the dsRNA-dependent protein kinase (PKR) may be responsible for these changes in protein synthesis and degradation. In order to evaluate whether this is also applicable to cancer cachexia the action of a PKR inhibitor on the development of cachexia has been studied in mice bearing the MAC16 tumour. Treatment of animals with the PKR inhibitor (5 mg kg-1) significantly reduced levels of phospho-PKR in muscle down to that found in non-tumour-bearing mice, and effectively attenuated the depression of body weight, with increased muscle mass, and also inhibited tumour growth. There was an increase in protein synthesis in skeletal muscle, which paralleled a decrease in eukaryotic initiation factor 2α phosphorylation. Protein degradation rates in skeletal muscle were also significantly decreased, as was proteasome activity levels and expression. Myosin levels were increased up to values found in non-tumour-bearing animals. Proteasome expression correlated with a decreased nuclear accumulation of nuclear factor-κB (NF-κB). The PKR inhibitor also significantly inhibited tumour growth, although this appeared to be a separate event from the effect on muscle wasting. These results suggest that inhibition of the autophosphorylation of PKR may represent an appropriate target for the attenuation of muscle atrophy in cancer cachexia. © 2007 Cancer Research UK.

Effect of zinc-alpha 2-glycoprotein (ZAG) on expression of uncoupling proteins in skeletal muscle and adipose tissue

The plasma protein zinc-α2-glycoprotein (ZAG) has been shown to be identical with a lipid mobilizing factor capable of inducing loss of adipose tissue in cancer cachexia through an increased lipid mobilization and utilization. The ability of ZAG to induce uncoupling protein (UCP) expression has been determined using in vitro models of adipose tissue and skeletal muscle. ZAG induced a concentration-dependent increase in the expression of UCP-1 in primary cultures of brown, but not white, adipose tissue, and this effect was attenuated by the β3-adrenergic receptor (β3-AR) antagonist SR59230A. A 6.5-fold increase in UCP-1 expression was found in brown adipose tissue after incubation with 0.58 μM ZAG. ZAG also increased UCP-2 expression 3.5-fold in C2C12 murine myotubes, and this effect was also attenuated by SR59230A and potentiated by isobutylmethylxanthine, suggesting a cyclic AMP-mediated process through interaction with a β3-AR. ZAG also produced a dose-dependent increase in UCP-3 in murine myotubes with a 2.5-fold increase at 0.58 μM ZAG. This effect was not mediated through the β3-AR, but instead appeared to require mitogen activated protein kinase. These results confirm the ability of ZAG to directly influence UCP expression, which may play an important role in lipid utilization during cancer cachexia. © 2004 Elsevier Ireland Ltd. All rights reserved.

The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport

The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. A shared feature of fab1Delta yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P(2), in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.

Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells:role of ERK1/2 in H2O2-induced cell death

Reactive oxygen species including H2O2 activate an array of intracellular signalling cascades that are closely associated with cell death and cell survival pathways. The human neuroblastoma SH-SY5Y cell line is widely used as model cell system for studying neuronal cell death induced by oxidative stress. However, at present very little is known about the signalling pathways activated by H2O2 in SH-SY5Y cells. Therefore, in this study we have investigated the effect of H2(O2 on extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (PKB) activation in undifferentiated and differentiated SH-SY5Y cells. H2O2 stimulated time and concentration increases in ERK1/2, JNK and PKB phosphorylation in undifferentiated and differentiated SH-SY5Y cells. No increases in p38 MAPK phosphorylation were observed following H2O2 treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 ((2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited H2O2-induced increases in ERK1/2 and PKB phosphorylation. Furthermore, H2O2-mediated increases in ERK1/2 activation were sensitive to the MAPK kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK responses were blocked by the JNK inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Treatment of SH-SY5Y cells with H2O2 (1 mM; 16 h) significantly increased the release of lactate dehydrogenase (LDH) into the culture medium indicative of a decrease in cell viability. Pre-treatment with wortmannin, SP 600125 or SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; p38 MAPK inhibitor) had no effect on H2O2-induced LDH release from undifferentiated or differentiated SH-SY5Y cells. In contrast, PD 98059 and LY 294002 significantly decreased H2O2-induced cell death in both undifferentiated and differentiated SH-SY5Y cells. In conclusion, we have shown that H2O2 stimulates robust increases in ERK1/2, JNK and PKB in undifferentiated and differentiated SH-SY5Y cells. Furthermore, the data presented clearly suggest that inhibition of the ERK1/2 pathway protects SH-SY5Y cells from H2O2-induced cell death.

Angiopoietin-1 induces migration of monocytes in a tie-2 and integrin-independent manner

Angiopoietin-1 (Ang-1) is an angiogenic growth factor that activates Tie-2 and integrins to promote vessel wall remodeling. The recent finding of the potential proatherogenic effects of Ang-1 prompted us to investigate whether Ang-1 promotes monocyte chemotaxis, endothelial binding, and transendothelial migration, key events in the progression of atherosclerosis. Here, we show that Ang-1 induces chemotaxis of monocytes in a manner that is independent of Tie-2 and integrin binding but dependent on phosphoinositide 3-kinase and heparin. In addition, Ang-1 promoted phosphoinositide 3-kinase-dependent binding of monocytes to endothelial monolayers and stimulated transendothelial migration. Fluorescence-activated cell sorting analysis showed that exogenous Ang-1 adheres directly to monocytes as well as to human umbilical endothelial cells, but neither Tie-2 mRNA nor protein were expressed by primary monocytes. Although Ang-1 binding to human umbilical endothelial cells was partially Tie-2 and integrin dependent, Ang-1 binding to monocytes was independent of these factors. Finally, preincubation of monocytes with soluble heparin abrogated Ang-1 binding to monocytes and migration, and partially prevented Ang-1 binding to human umbilical endothelial cells. In summary, Ang-1 induces chemotaxis of monocytes by a mechanism that is dependent on phosphoinositide 3-kinase and heparin but independent of Tie-2 and integrins. The ability of Ang-1 to recruit monocytes suggests it may play a role in inflammatory angiogenesis and may promote atherosclerosis.

Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells

The proteinase-activated receptor 2 (PAR-2) expression is increased in endothelial cells derived from women with preeclampsia, characterized by widespread maternal endothelial damage, which occurs as a consequence of elevated soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; commonly known as sFlt-1) in the maternal circulation. Because PAR-2 is upregulated by proinflammatory cytokines and activated by blood coagulation serine proteinases, we investigated whether activation of PAR-2 contributed to sVEGFR-1 release. PAR-2–activating peptides (SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2) and factor Xa increased the expression and release of sVEGFR-1 from human umbilical vein endothelial cells. Enzyme-specific, dominant-negative mutants and small interfering RNA were used to demonstrate that PAR-2–mediated sVEGFR-1 release depended on protein kinase C-ß1 and protein kinase C-e, which required intracellular transactivation of epidermal growth factor receptor 1, leading to mitogen-activated protein kinase activation. Overexpression of heme oxygenase 1 and its gaseous product, carbon monoxide, decreased PAR-2–stimulated sVEGFR-1 release from human umbilical vein endothelial cells. Simvastatin, which upregulates heme oxygenase 1, also suppressed PAR-2–mediated sVEGFR-1 release. These results show that endothelial PAR-2 activation leading to increased sVEGFR-1 release may contribute to the maternal vascular dysfunction observed in preeclampsia and highlights the PAR-2 pathway as a potential therapeutic target for the treatment of preeclampsia.

Deficiency in catechol-O-methyltransferase and 2-methoxyoestradiol is associated with pre-eclampsia

Despite intense investigation, mechanisms that facilitate the emergence of the pre-eclampsia phenotype in women are still unknown. Placental hypoxia, hypertension, proteinuria and oedema are the principal clinical features of this disease. It is speculated that hypoxia-driven disruption of the angiogenic balance involving vascular endothelial growth factor (VEGF)/placenta-derived growth factor (PLGF) and soluble Fms-like tyrosine kinase-1 (sFLT-1, the soluble form of VEGF receptor 1) might contribute to some of the maternal symptoms of pre-eclampsia. However, pre-eclampsia does not develop in all women with high sFLT-1 or low PLGF levels, and it also occurs in some women with low sFLT-1 and high PLGF levels. Moreover, recent experiments strongly suggest that several soluble factors affecting the vasculature are probably elevated because of placental hypoxia in the pre-eclamptic women, indicating that upstream molecular defect(s) may contribute to pre-eclampsia. Here we show that pregnant mice deficient in catechol-O-methyltransferase (COMT) show a pre-eclampsia-like phenotype resulting from an absence of 2-methoxyoestradiol (2-ME), a natural metabolite of oestradiol that is elevated during the third trimester of normal human pregnancy. 2-ME ameliorates all pre-eclampsia-like features without toxicity in the Comt(-/-) pregnant mice and suppresses placental hypoxia, hypoxia-inducible factor-1alpha expression and sFLT-1 elevation. The levels of COMT and 2-ME are significantly lower in women with severe pre-eclampsia. Our studies identify a genetic mouse model for pre-eclampsia and suggest that 2-ME may have utility as a plasma and urine diagnostic marker for this disease, and may also serve as a therapeutic supplement to prevent or treat this disorder.

Sequence and chemistry requirements for a novel aptameric oligonucleotide inhibitor of EGF receptor tyrosine kinase activity

We have previously identified a phosphorothioate oligonucleotide (PS-ODN) that inhibited epidermal growth factor receptor tyrosine kinase (TK) activity both in cell fractions and in intact A431 cells. Since ODN-based TK inhibitors may have anti-cancer applications and may also help understand the non-antisense mediated effects of PS-ODNs, we have further studied the sequence and chemistry requirements of the parent PS-ODN (sequence: 5′-GGA GGG TCG CAT CGC-3′) as a sequence-dependent TK inhibitor. Sequence deletion and substitution studies revealed that the 5′-terminal GGA GGG hexamer sequence in the parent compound was essential for anti-TK activity in A431 cells. Site-specific substitution of any G with a T in this 5′-terminal motif within the parent compound caused a significant loss in anti-TK activity. The fully PS-modified hexameric motif alone exhibited equipotent activity as the parent 15-mer whereas phosphodiester (PO) or 2′-O-methyl-modified versions of this motif had significantly reduced anti-TK activity. Further, T substitutions within the two 5′-terminal G residues of the hexameric PS-ODN to produce a sequence, TTA GGG, representing the telomeric repeats in human chromosomes, also did not exhibit a significant anti-TK activity. Multiple repeats of the active hexameric motif in PS-ODNs resulted in more potent inhibitors of TK activity than the parent ODN. These results suggested that PS-ODNs, but not PO or 2′-O-methyl modified ODNs, containing the GGA GGG motif can exert potent anti-TK activity which may be desirable in some anti-tumor applications. Additionally, the presence of this previously unidentified motif in antisense PS-ODN constructs may contribute to their biological effects in vitro and in vivo and should be accounted for in the design of the PS-modified antisense ODNs. © 2002 Published by Elsevier Science Inc.

Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast

Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.

Mechanism of attenuation of protein loss in murine C2C12 myotubes by d-myo-inositol 1,2,6-triphosphate

d-Myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrophy in a murine cachexia model through an increase in protein synthesis and a decrease in degradation. The mechanism of this effect has been investigated in murine myotubes using a range of catabolic stimuli, including proteolysis-inducing factor (PIF), angiotensin II (Ang II), lipopolysaccharide, and tumor necrosis factor-α/interferon-γ. At a concentration of 100 μM AT was found to attenuate both the induction of protein degradation and depression of protein synthesis in response to all stimuli. The effect on protein degradation was accompanied by attenuation of the increased expression and activity of the ubiquitin-proteasome pathway. This suggests that AT inhibits a signalling step common to all four agents. This target has been shown to be activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) and the subsequent phosphorylation of eukaryotic initiation factor 2 on the α-subunit, together with downstream signalling pathways leading to protein degradation. AT also inhibited activation of caspase-3/-8, which is thought to lead to activation of PKR. The mechanism of this effect may be related to the ability of AT to chelate divalent metal ions, since the attenuation of the increased activity of the ubiquitin-proteasome pathway by PIF and Ang II, as well as the depression of protein synthesis by PIF, were reversed by increasing concentrations of Zn2+. The ability of AT to attenuate muscle atrophy by a range of stimuli suggests that it may be effective in several catabolic conditions. © 2009 Elsevier Inc. All rights reserved.

Receptor for activated C Kinase 1 protein binds to and activates the human estrogen receptor α

The classical concept of estrogen receptor (ER) activation is that steroid passes the cell membrane, binds to its specific protein receptor in the cell's cytoplasm and the steroid-receptor complex travels to the nucleus where it activates responsive genes. This basic idea has been challenged by results of experiments demonstrating insulin-like growth factor 1 (IGF-1) activation of the ER in the complete absence of estrogen suggesting at least one other mechanism of ER activation not involving steroid. One explanation is that activation of the cell surface IGF-1 receptor leads to synthesis of an intracellular protein(s) able to bind to and stimulate the ER. Based on results using the two-hybrid system, coimmunoprecipitation and transfection-luciferase assays, we herein show that one of these proteins could well be receptor for activated C kinase 1 (RACK-1). Using the human ER type α (ER-α) as bait, a cloned complementary deoxyribonucleic acid (cDNA) library from IGF-1 treated human breast cancer MCF-7 cells was screened for ER-α - protein interactions. Many positive clones were obtained which contained the RACK-1 cDNA sequence. Coimmunoprecipitation of in-vitro translation products of the ER-α and RACK-1 confirmed the interaction between the two proteins. Transfection studies using the estrogen response element spliced to a luciferase reporter gene revealed that constitutive RACK-1 expression was able to powerfully stimulate ER-α activity under estrogen-free conditions. This effect could be enhanced by 17β-estradiol (E2) and blocked by tamoxifen, an E2 antagonist. These results show that RACK-1 is able to activate the ER-α in the absence of E2, although together with the latter, enhanced effects occur. Since RACK-1 gene expression is stimulated by IGF-1, it is distinctly possible that RACK-1 is the mediator of the stimulatory effects of IGF-1 on ER-α. © 2014 JMS.

The hypotensive effect of acute and chronic AMP-activated protein kinase activation in normal and hyperlipidemic mice

AMP-activated protein kinase (AMPK) is present in the arterial wall and is activated in response to cellular stressors that raise AMP relative to ADP/ATP. Activation of AMPK in vivo lowers blood pressure but the influence of hyperlipidemia on this response has not been studied. ApoE-/- mice on high fat diet for 6 weeks and age-matched controls were treated with the AMPK activator, AICAR daily for two weeks. Under anesthesia, the carotid artery was cannulated for blood pressure measurements. Aortic tissue was removed for in vitro functional experiments and AMPK activity was measured in artery homogenates by Western blotting. ApoE-/- mice had significantly raised mean arterial pressure; chronic AICAR treatment normalized this but had no effect in normolipidemic mice, whereas acute administration of AICAR lowered mean arterial pressure in both groups. Chronic AICAR treatment increased phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase in normolipidemic but not ApoE-/- mice. In aortic rings, AMPK activation induced vasodilation and an anticontractile effect, which was attenuated in ApoE-/- mice. This study demonstrates that hyperlipidemia dysregulates the AMPK pathway in the arterial wall but this effect can be reversed by AMPK activation, possibly through improving vessel compliance.

Drug repurposing screen identifies Foxo1-dependent angiopoietin-2 regulation in sepsis

Objective: The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leakage and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. Design: Laboratory and animal research plus prospective placebo-controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). Setting: Research laboratories of Hannover Medical School and Harvard Medical School. Patients: Septic patients/C57Bl/6 mice and human endothelial cells. Interventions: Food and Drug Administration-approved library screening. Measurements and Main Results: In a cell-based screen of more than 650 Food and Drug Administration-approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. Conclusions: 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2's dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage.

Negative feedback regulation of the ERK1/2 MAPK pathway

The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signalling pathway regulates many cellular functions, including proliferation, differentiation, and transformation. To reliably convert external stimuli into specific cellular responses and to adapt to environmental circumstances, the pathway must be integrated into the overall signalling activity of the cell. Multiple mechanisms have evolved to perform this role. In this review, we will focus on negative feedback mechanisms and examine how they shape ERK1/2 MAPK signalling. We will first discuss the extensive number of negative feedback loops targeting the different components of the ERK1/2 MAPK cascade, specifically the direct posttranslational modification of pathway components by downstream protein kinases and the induction of de novo gene synthesis of specific pathway inhibitors. We will then evaluate how negative feedback modulates the spatiotemporal signalling dynamics of the ERK1/2 pathway regarding signalling amplitude and duration as well as subcellular localisation. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway thus represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. We will, therefore, discuss the effect of ERK1/2 MAPK feedback regulation on cancer treatment and how it contributes to reduced clinical efficacy of therapeutic agents and the development of drug resistance.