Statins (3-hydroxy-3-methylglutaryl-CoA [HMG-CoA] reductase inhibitors) inhibit the rate-limiting step in the mevalonate pathway, conversion of HMG-CoA to mevalonate, by competitive inhibition with the enzyme HMG-CoA reductase. Statins not only lower cholesterol levels, but are also thought to exert neuroprotective and neurogenic effects that may be beneficial in treating brain and spinal cord injuries. Data presented here illustrate that simvastatin enables neurite outgrowth in the presence of growth-inhibitory molecules commonly found at central nervous system (CNS) injury sites. To assess the effect of simvastatin on neurite outgrowth in the presence of inhibitory molecules present at CNS injury sites, rat embryonic cortex explants or postnatal spinal cord explants were grown on membrane filters prepared with alternating stripes of laminin and myelin/laminin. Immunostaining indicated that myelin stripes contain myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and Nogo, but do not contain chondroitin sulfate proteoglycan (CSPG). When control explants were grown in the presence of alternating stripes, neurite outgrowth preferentially extended in regions containing laminin only. In contrast, neurite outgrowth from explants grown in the presence of simvastatin was significantly less selective for laminin regions and was able to extend into regions containing myelin (p < 0.01). Simvastatin-induced effects were reversed by addition of mevalonate. Isoprenyl transferase inhibitors GGTI-286 and FTI-277, inhibitors of biochemical steps subsequent to HMG-CoA conversion to mevalonate, mimicked simvastatin- induced effects. These data suggest that simvastatin counteracts myelin-associated neurite outgrowth inhibition signals via mevalonate pathway inhibition, and may be beneficial in promoting axon regeneration in brain and spinal cord injury.

Farnesyltransferase inhibitors (FTIs) represent a new class of anticancer drugs that show promise in blocking the growth of tumors. Here, we report that FTIs are capable of inducing apoptosis of transformed but not untransformed cells. Treatment of v-K-ras-transformed normal rat kidney (KNRK) cells with FTIs leads to the induction of apoptotic cell morphology, chromatin condensation and DNA fragmentation. In addition, fluorescence-activated cell sorter analysis of FTI-treated KNRK cells shows a sub-G1 apoptotic peak (chromosome content of <2 N). This FTI-induced apoptosis is evident only when the cells are grown in low serum conditions (0.1% fetal calf serum) and is observed selectively with transformed KNRK cells and not with untransformed NRK cells. Further analysis of the mechanism underlying this apoptosis has shown that FTI treatment of KNRK cells results in the activation of caspase 3 but not caspase 1. Moreover, the addition of Z-DEVD-fmk, an agent that interferes with caspase 3 activity, can inhibit FTI-induced apoptosis in a dose-dependent manner. Introduction of the CASP-3 gene into MCF7 cells, which lack caspase 3 activity, results in a significant increase of FTI-induced apoptosis. Furthermore, FTI induces the release of cytochrome c into the cytosol. This release is an important feature of caspase 3-mediated apoptosis. These results suggest that FTIs induce apoptosis through the release of cytochrome c from the mitochondria resulting in caspase 3 activation.

A series of protein farnesyltransferase inhibitor ester prodrugs of FTI-2148 (17) were synthesized in order to evaluate the effects of ester structure modification on antimalarial activity and for further development of a farnesyltransferase inhibitor with in vivo activity. Evaluation against P. falciparum in red blood cells showed that all the investigated esters exhibited significant antimalarial activity, with the benzyl ester 16 showing the best inhibition (ED50=150 nM). Additionally, compound 16 displayed in vivo activity and was found to suppress parasitemia by 46.1% at a dose of 50 mg kg(-1) day(-1) against Plasmodium berghei in mice. The enhanced inhibition potency of the esters is consistent with improved cell membrane permeability compared to that of the free acid. The results of this study suggest that protein farnesyltransferase is a valid antimalarial drug target and that the antimalarial activity of these compounds derives from a balance between the hydrophobic character and the size and conformation of the ester moiety.

The efficacy of tamoxifen in the hormonal therapy of breast cancer is well established, but therapeutic resistance is inevitable. FTIs are a new class of anticancer drugs that are in phase III clinical evaluation. Since the mechanisms of action of these 2 classes of drugs are different, we tested the combination of tamoxifen and FTI-277 on inhibiting proliferation of hormone-dependent MCF-7 human breast cancer cells. An additive effect on cell proliferation was demonstrated, accompanied by an additive G(0)/G(1) arrest. The major effect of the combination of the 2 drugs was to maintain p21(waf/cip1) at an intermediate level, higher than that observed in the presence of tamoxifen alone. This was associated with an additive effect on inactivation of cyclin E-Cdk2 complexes and decreased phosphorylation of pRb and p130 pocket proteins. These effects were accompanied by increased association of 2 CDIs, p27(kip1) and p21(waf/cip1), with cyclin E-Cdk2 complexes. These data demonstrate that the additive effect is likely predominantly due to the recruitment of p27(kip1) and, to a lesser extent, p21(waf/cip1) into the cyclin E-Cdk2 complexes. Together, these results suggest that the combination of FTI and tamoxifen may increase the antitumor effect of either drug alone in breast cancer.

Ras protein plays pivotal roles in control of normal and transformed cell growth. These ras genes are mutated in 30% of human cancer. For functioning of Ras protein its prenylation (farnesylation) is required. Therefore targeting Ras farnesylation is valuable approach for cancer treatment. Farnesyltransferase inhibitor (FTI) has been developed as anticancer drug and is currently evaluated in clinical trials. Different types of FTI have been identified that inhibit Ras farnesylation. FTI in combination with some cytotoxic antineoplastic drugs, exhibit additive effects.

In order to identify small G protein (s) which contributes to the proliferation of vascular smooth muscle cells (VSMCs), we examined the effect of an HMG-CoA reductase inhibitor (cerivastatin), a farnesyltransferase inhibitor (FTI-277), a geranyl geranyl transferase inhibitor (GGTI-286) and a Rho kinase inhibitor (Y-27632) on the proliferation of cultured rat VSMCs stimulated with 20ng/ml platelet-derived growth factor (PDGF)-BB. Cerivastatin and GGTI-286, but not FTI-277, suppressed the PDGF-BB-induced activation of extracellular signal related kinase (ERK1/2). The inhibitory effect of cerivastatin on the PDGF-BB-induced activation of ERK1/2 was fully recovered by the addition of geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP). Cerivastatin and GGTI-286, but not FTI-277, suppressed the PDGF-BB-induced [3H] thymidine incorporation and activation of ornitine decarboxylase (ODC), both of which were fully recovered by the addition of GGPP, but not FPP. These data indicate that the PDGF-BB-induced activation of ERK1/2 and proliferation of VSMCs depend upon geranylgeranylated small G protein. Immunoblotting analysis revealed the upregulation of Rho A protein in the membrane fractions of VSMCs stimulated by PDGF-BB. Furthermore, Y-27632 suppressed the PDGF-BB-induced activation of ERK1/2 and proliferation of VSMCs. On the basis of these data, we conclude that PDGF-BB stimulates the proliferation of VSMCs via the activation of Rho A. Rho kinase plays an important role in this process as an effector of Rho A.

The cytoplasmic deacetylase HDAC6 is an important regulator of cellular pathways that include response to stress, protein folding, microtubule stability, and cell migration, thus representing an attractive target for cancer chemotherapy. However, little is known about its upstream regulation. Our previous work has implicated HDAC6 as a new protein target for the farnesyltransferase inhibitors (FTIs), although HDAC6 lacks a farnesylation motif. Here we show that the protein farnesyltransferase (FTase) and HDAC6 are present in a protein complex together with microtubules in vivo and in vitro. FTase binds microtubules directly via its alpha subunit, and this association requires the C terminus of tubulin. Treatment with an FTI removed FTase, but not HDAC6, from the protein complex, suggesting that the active form of FTase is bound to microtubules. Importantly, the removal of FTase from microtubules abrogated HDAC6 activity, as did a stable knockdown of the alpha subunit of FTase (FTalphaKD). Interestingly, the FTalphaKD cells showed increased sensitivity to the antiproliferative effects of Taxol and the FTI lonafarnib when used either as single agents or in combination as compared with parental cells. Altogether, these data suggest that FTase, via its tubulin-association, is a critical upstream regulator of HDAC6 activity and that FTase expression could help stratify cancer patients that would most benefit from this treatment.

Hepatic hydroxymethyl glutary coenzyme A HMG-CoA reductase inhibitors (statins) have various anti atherosclerosis pleiotropic effects that are independent of cholesterol reduction. Human serum paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL) and inhibits the oxidative modification of low-density lipoprotein (LDL). We investigated the effects of statins on PON1 gene transcription using a reporter gene assay. Promoter activity of the PON1 gene was estimated by measuring luciferase activity of plasmids with a PON1 promoter region transfected into human hepatoma HepG2 cells and human embryonic kidney (HEK) 293 cells. Pitavastatin, simvastatin, and atorvastatin each significantly increased PON1 promoter activity, and the transactivation by pitavastatin was abrogated by mevalonic acid and farnesyl pyrophosphate (FPP), however, not by geranylgeranyl pyrophosphate. Further, PON1 promoter activity was enhanced by farnesyl transferase inhibitor (FTI), but not by geranylgeranyl transferase inhibitor (GGTI). PON1 gene transcription has been reported to be dependent on Sp1 and the transactivation by pitavastatin was completely abrogated by mithramycin, an inhibitor of Sp1. Our results suggest that pitavastatin activates transcription of the PON1 gene through the FPP pathway, which may play an important role in the anti atherosclerotic effects of statins.

Three-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, known as statins, induce skeletal muscle injury including myalgia, myositis, and rhabdomyolysis. The mechanism of this myotoxicity remains unknown. This study examined the effect of statins on single skeletal myofibers enzymatically isolated from the rat flexor digitorum brevis muscles. Fluvastatin and pravastatin induced the formation of numerous vacuoles in the myofibers after 72 h of treatment. This effect progressed in a time- and concentration-dependent manner and, consequently, cell death occurred after 120 h. Electron micrographs revealed craters along the sarcolemma and swelling of the sarcoplasmic reticula and mitochondria, in addition to intracellular vacuoles. When caffeine was added after 72 h of fluvastatin treatment, contractile shortening of statin-treated myofibers was significantly attenuated and blebs formed on the surface of the myofibers. The coapplication of geranylgeranylpyrophosphate (GGPP) with fluvastatin prevented the morphological changes, while that of farnesylpyrophosphate (FPP) was ineffective. Furthermore, perillyl alcohol, an inhibitor of Rab geranylgeranyl transferase and geranylgeranyl transferase-I (GGTase-I), mimicked the effect of statins, while a specific GGTase-I inhibitor (GGTI-298) or a farnesyl transferase inhibitor (FTI-277) failed to do so. These results suggest that the inactivation of Rab GTPase, which involved in intracellular membrane transport, is a crucial factor in statin-induced-morphological abnormality in skeletal muscle fibers.

Post-translational modification of proteins by the addition of a farnesyl group is critical for the function of a number of proteins involved in signal transduction. Farnesylation facilitates their membrane association and also promotes protein-protein interaction. Recently, progress has been made in understanding the biological significance of farnesylation. First, effects of farnesyltransferase inhibitors (FTIs) on cancer cells have been examined using a variety of human cancer cells. This study showed that one of the major effects of FTIs is to alter cell cycle progression. Both G0/G1 enrichment and G2/M accumulation were observed depending on the cell line examined. Second, a number of novel farnesylated proteins have been characterized. Of these, Rheb and CENP-E,F are of particular interest. Rheb, a novel member of the Ras superfamily G-proteins, may play a role in the G1 phase of the cell cycle. CENP-E,F are centromere associated motors that play critical roles in mitosis. These results suggest important contributions of farnesylated proteins in the regulation of cell cycle progression.

Maternal hypothyroidism may be associated with a variety of pregnancy complications.

OBJECTIVE

We have evaluated the perinatal consequences of maternal hypothyroidism in early and late gestation.

METHODS

Retrospective study of pregnant women, with quasi-experimental design comparing different subjects.

METHODS

Forty-three pregnancies in 42 women with hypothyroidism--either biochemically hypothyroid or with a history of hypothyroidism on adequate replacement--and no other preexisting medical conditions.

METHODS

Free thyroxine index (FTI), TSH, and haematocrit at initial antepartum presentation and near term gestation. Pregnancy outcome variables including: rate of Caesarean section performed for fetal distress in labour, neonatal weight percentile, and gestational age at birth.

RESULTS

Of 42 hypothyroid pregnancies, six were complicated by fetal distress in labour leading to Caesarean section. Five of these six were severely hypothyroid (defined as FTI < or = 0.6 (normal range 1.1-4.4)) on initial antepartum presentation. In contrast, of the 36 pregnancies without fetal distress, only four initially presented severely hypothyroid (P < 0.001). Conversely, 56% (5/9) of pregnancies which initially presented severely hypothyroid were subsequently complicated by Caesarean section for fetal distress in labour. This compared to 3% (1/33) among those who presented either mildly hypothyroid or euthyroid on replacement (P < 0.0001). Fetal distress correlated with low FTI (P < 0.001) and high TSH at initial presentation. However, it was independent of FTI near term. A relation between fetal distress and TSH near term did not reach statistical significance. Fetal distress also correlated with low maternal haematocrit on admission to labour and delivery (P < 0.05), but was independent of haematocrit and gestational age at initial presentation, neonatal weight percentile, and gestational age at birth.

CONCLUSIONS

Severe maternal hypothyroidism early in gestation is strongly associated with fetal distress in labour. This suggests that (1) inadequate maternal replacement leads to fetal distress and (2) maternal thyroid status in early gestation may exert irreversible effects on the fetus, the placenta, and/or on subsequent maternal adaptations to pregnancy. Early adequate replacement therapy is especially prudent in pregnant women presenting with severe hypothyroidism.

To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibit malignant growth, or induce apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyltransferase (FTI), the enzyme most often responsible for prenylating Ras, showed clinical promise, but development of FTIs such as tipifarnib has been stalled by uncertainty about their mechanism of action, because Ras seemed unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Our initial observations of varied response by cancer cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using various tumor models. We describe our novel counterintuitive finding that endogenous Ras activity increases in cancer cell lines with low endogenous Ras activity when farnesyltransferase is inhibited by either tipifarnib or short hairpin RNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated extracellular signal–regulated kinase (ERK) and p38 mitogenactivated protein kinase (MAPK). Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Because Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interacts differently with downstream effector proteins in sensitive cancer cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition [corrected].

Ras activation is frequently observed in multiple myeloma either by mutation or through interleukin-6 receptor signaling. Recently, drugs designed to inhibit Ras have shown promise in preclinical myeloma models and in clinical trials. In this report, we characterize the pathways by which the clinically tested farnesyl transferase inhibitor (FTI) R115777 induces apoptosis in multiple myeloma cells. Contrary to the proposed mechanistic action of FTIs, we found that R115777 induces cell death despite Ras prenylation implying participation of Ras-independent mechanism(s). Apoptosis proceeded via an intrinsic cascade and was associated with an increase in the expression and activity of Bax. Bax activation correlated with a loss of mitochondrial membrane integrity and activation of the endoplasmic reticulum (ER) stress response. These pathways activate caspase-9 and consistent with this, cell death was prevented by caspase-9 blockade. Interestingly, cells overexpressing Bcl-X(L) remained partially sensitive to R115777 despite suppression of mitochondrial membrane dysfunction and ER-related stress. Taken together, these results indicate that R115777 induces apoptosis in a Ras-independent fashion via multiple intrinsic pathways.

BACKGROUND

For the diagnosis of thyroid disease, measurement of "free hormone" is generally accepted as an appropriate measure. However, valid assays measuring the free fraction of thyroxine (FT4) ideally must perform without bias, despite large variations in the concentrations and affinities of serum T4-binding proteins in the population. Several approaches have been taken to overcome such bias, and these have created considerable controversy in the field over the past decade.

METHODS

This review, from both a historical and an analytical standpoint, charts the progress made over more than 30 years in improvements to the performance of assays in common use for the measurement of FT4 in serum or plasma. It reexamines the theory behind early approaches to such assays [for example, the free thyroxine index (FTI) method], that preceded more accurate, two-step immunoassays or one-step analog techniques. It evaluates the continuous refinements to the latter assays that by now have largely supplanted the FTI approach and where the deficiencies that so exercised clinical chemists in the past have been virtually eliminated in the leading assays.

BACKGROUND

The basic Mass Action theory underpinning all such methods is discussed by assessing how far each particular approach obeys the criteria the theory imposes. In this, it is not the intention of the review to dissect individual commercial or academic assays, but rather to give guidance where appropriate as to how any assay said to measure FT4 can be conveniently evaluated by those intending to use it. Examples are given where inappropriate tests may wrongly imply assay invalidity by misinterpreting how FT4 assays work.

CONCLUSIONS

Detailed knowledge of the underlying theory is essential when devising tests for direct FT4 assays, to ensure that such tests do not overstep the practical limits of assay validity.

Thyroid function tests were measured in 645 patients admitted to an acute psychiatric disorders unit. Thirty-three percent had elevated serum thyroxine (T4), and 18 percent had an elevated free T4 index (FTI). Serum triiodothyronine (T3) was low, normal, or minimally elevated in 77 patients, with a high initial free T4 index. Twenty-two patients with an initial elevation of their free T4 index were serially followed (study group). Serum T4, free T4 index, and free T4 fell in every patient: serum T4 from 13.95 +/- 1.93 micrograms/dl (mean +/- standard deviation: SD) to 9.33 +/- 2.4 micrograms/dl (p less than 0.001); free T4 index, from 6.15 +/- 0.83 to 3.79 +/- 1.1 (p less than 0.001); free T4, from 2.43 +/- 0.65 mg/dl to 1.38 +/- 0.35 ng/dl (p less than 0.001). Serum T3 was initially normal or low, and then fell in 17 patients, and rose in five. Serial testing of thyrotropin-releasing hormone (TRH) demonstrated both flat and normal responses in patients with a variety of psychiatric diagnoses and at varying stages of thyroid disease activity.

Oncogenic RAS alleles encode proteins that accumulate in the guanosine triphosphate (GTP)-bound state. Because post-translational processing of Ras by farnesyltransferase is essential for biologic function, inhibitors of this enzyme have been developed as rational cancer therapeutics. We have investigated farnesyltransferase inhibitor (FTI) L-744,832 in an in vivo murine model of myeloid leukemia that is associated with inactivation of the Nf1 tumor suppressor gene. Nf1 encodes a GTPase activating protein for Ras, and Nf1-deficient (Nf1-/-) hematopoietic cells show hyperactive Ras signaling through the mitogen-activated protein (MAP) kinase pathway. L-744,832 inhibited H-Ras prenylation in cell lines and in primary hematopoietic cells and abrogated the in vitro growth of myeloid progenitor colonies in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). This FTI also partially blocked GM-CSF-induced MAP kinase activation, but did not reduce constitutively elevated levels of MAP kinase activity in primary Nf1-/- cells. Injection of a single dose of 40 or 80 mg/kg of L-744, 832 increased the amount of unprocessed H-Ras in bone marrow cells, but had no detectable effect on N-Ras. Adoptive transfer of Nf1-/- hematopoietic cells into irradiated mice induces a myeloproliferative disorder that did not respond to L-744,832 treatment. We speculate that the lack of efficacy in this model is due to the resistance of N-Ras and K-Ras processing to inhibition by this FTI.

Smooth muscle cells promote fibroproliferative airway remodeling in asthma, and transforming growth factor β1 (TGFβ1) is a key inductive signal. Statins are widely used to treat hyperlipidemia. Growing evidence indicates they also exert a positive impact on lung health, but the underlying mechanisms are unclear. We assessed the effects of 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase inhibition with simvastatin on the fibrotic function of primary cultured human airway smooth muscle cells. Simvastatin blocked de novo cholesterol synthesis, but total myocyte cholesterol content was unaffected. Simvastatin also abrogated TGFβ1-induced collagen I and fibronectin expression, and prevented collagen I secretion. The depletion of mevalonate cascade intermediates downstream from HMG-CoA underpinned the effects of simvastatin, because co-incubation with mevalonate, geranylgeranylpyrophosphate, or farnesylpyrophosphate prevented the inhibition of matrix protein expression. We also showed that human airway myocytes express both geranylgeranyl transferase 1 (GGT1) and farnesyltransferase (FT), and the inhibition of GGT1 (GGTI inhibitor-286, 10 μM), but not FT (FTI inhibitor-277, 10 μM), mirrored the suppressive effects of simvastatin on collagen I and fibronectin expression and collagen I secretion. Moreover, simvastatin and GGTI-286 both prevented TGFβ1-induced membrane association of RhoA, a downstream target of GGT1. Our findings suggest that simvastatin and GGTI-286 inhibit synthesis and secretion of extracellular matrix proteins by human airway smooth muscle cells by suppressing GGT1-mediated posttranslational modification of signaling molecules such as RhoA. These findings reveal mechanisms related to evidence for the positive impact of statins on pulmonary health.

Human Brox is a newly identified 46 kDa protein that has a Bro1 domain-like sequence and a C-terminal thioester-linkage site of isoprenoid lipid (CAAX motif) (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid). Mammalian Alix and its yeast ortholog, Bro1, are known to associate with charged multivesicular body protein 4 (CHMP4), a component of endosomal sorting complex required for transport III, via their Bro1 domains and to play roles in sorting of ubiquitinated cargoes. We investigated whether Brox has an authentic Bro1 domain on the basis of its capacity for interacting with CHMP4s. Both Strep Tactin binding sequence (Strep)-tagged wild-type Brox (Strep-Brox(WT)) and Strep-tagged farnesylation-defective mutant (Cys->>Ser mutation; Strep-Brox(C408S)) pulled down FLAG-tagged CHMP4b that was coexpressed in HEK293 cells. Treatment of cells with a farnesyltransferase inhibitor, FTI-277, caused an electrophoretic mobility shift of Strep-Brox(WT), and the mobility coincided with that of Strep-Brox(C408S). The inhibitor also caused a mobility shift of endogenous Brox detected by western blotting using polyclonal antibodies to Brox, suggesting farnesylation of Brox in vivo. Fluorescence microscopic analyses revealed that Strep-Brox(WT) exhibited accumulation in the perinuclear area and caused a punctate pattern of FLAG-CHMP4b that was constitutively expressed in HEK293 cells. On the other hand, Strep-Brox(C408S) showed a diffuse pattern throughout the cell, including the nucleus, and did not cause accumulation of FLAG-CHMP4b. Fluorescent signals of monomeric green fluorescent protein (mGFP)-fused Brox(WT) merged partly with those of Golgi markers and with those of abnormal endosomes induced by overexpression of a dominant negative mutant of AAA type ATPase SKD1/Vps4B in HeLa cells, but such colocalization was less efficient for mGFP-Brox(C408S). These results suggest a physiological significance of farnesylation of Brox in its subcellular distribution and efficient interaction with CHMP4s in vivo.

Focal adhesion kinase (FAK) is a widely expressed nonreceptor tyrosine kinase found in focal adhesions. FAK has been indicated as a point of convergence of other signaling pathways including platelet-derived growth factor (PDGF) receptors, and recently, FAK tyrosine phosphorylation has been shown to be stimulated by PDGF. In the present study we assessed the role of Ras as a possible intermediate protein regulating PDGF-induced FAK tyrosine phosphorylation in human hepatic stellate cells (HSCs), liver-specific pericytes primarily involved in the pathogenesis of liver fibrosis. For this purpose, cells were first subjected to retroviral-mediated gene transfer with a dominant-negative mutant of Ras (N17Ras). This resulted in a marked inhibition of PDGF-induced FAK tyrosine phosphorylation together with the expected reduction of PDGF-induced extracellular signal-regulated kinase activity (ERK). Afterward, the effects of pharmacological agents potentially affecting Ras isoprenylation were evaluated. PDGF-induced FAK tyrosine phosphorylation, ERK activity and intracellular calcium increase, as well as the biological effects of this growth factor, (i.e., mitogenesis and cell migration) were effectively blocked by GGTI-298, an inhibitor of geranylgeranyltransferase I. Inhibition of Ras processing obtained with FTI-277, an inhibitor of farnesyltransferase, resulted in detectable effects only at high doses. Taken together, these results establish that Ras operates as a protein-linking PDGF-beta receptor to FAK in human HSCs, and that signaling molecules requiring geranylgeranylation may also be involved in this process.

Novel strategies are required for treatment of acute myeloid leukaemia (AML) and higher risk myelodysplastic syndrome (MDS) patients who are not eligible for intensive chemotherapy and/or allogenic stem cell transplantation. As activating RAS mutations are frequent in these diseases, one novel approach, consisting of interfering with isoprenylation of RAS proteins by farnesyltransferase inhibitors (FTIs), has been proposed. Clinical phase II studies with the oral FTIs tipifarnib and lonafarnib in previously untreated AML, MDS and chronic myelomonocytic leukaemia yielded rather encouraging results while results in relapsed and/or refractory AML were disappointing. Results of a phase III trial in untreated AML in the elderly with tipifarnib were also disappointing. Clinical responses were not related to RAS mutations, suggesting additional actions of FTIs on other molecular targets. The combination of existing FTIs with other treatments, such as chemotherapy (in AML) and hypomethylating agents (in MDS and AML), is under investigation. Ongoing studies will also determine if gene profiling analysis may help to identify patients that will respond to FTIs.

The farnesyl transferase inhibitor (FTI) SCH66336 has been shown to have antitumor activities in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. However, its mechanism of action has not been well defined. Here, we report that the insulin-like growth factor (IGF) binding protein (IGFBP)-3 mediates antitumor activities of SCH66336 in HNSCC by inhibiting angiogenesis. SCH66336 significantly suppressed HNSCC tumor growth and angiogenesis via mechanisms that are independent of H-Ras and RhoB. By inducing IGFBP-3 secretion from HNSCC cells, this compound suppresses angiogenic activities of endothelial cells, including vessel formation in chorioallantoic membranes of chick, endothelial cell sprouting from chick aorta, and capillary tube formation of human umbilical vascular endothelial cells (HUVEC). Knockdown of IGFBP-3 expression in HNSCC cells by RNA interference or depletion of IGFBP-3 in HUVECs by neutralizing antibody effectively blocked the effects of IGFBP-3 secreted from SCH66336-treated HNSCC cells on HUVECs. These findings suggest that IGFBP-3 could be a primary target for antitumor activities of FTIs and that IGFBP-3 is an effective therapeutic approach against angiogenesis in HNSCC.

BMS-214662 is a potent and selective inhibitor of farnesyltransferase (FTI). In rodent fibroblasts transformed by oncogenes, BMS-214662 reversed the H-Ras-transformed phenotype but not that of K-Ras or other oncogenes. In soft agar growth assays, BMS-214662 showed good potency in inhibiting H-ras-transformed rodent cells, A2780 human ovarian carcinoma tumor cells, and HCT-116 human colon carcinoma tumor cells. Inhibition of H-Ras processing in HCT-116 human colon tumor cells was more rapid than in H-Ras-transformed rodent fibroblast tumors. BMS-214662 is the most potent apoptotic FTI known and demonstrated broad spectrum yet robust cell-selective cytotoxic activity against a panel of cell lines with diverse histology. The presence of a mutant ras oncogene was not a prerequisite for sensitivity. Athymic and conventional mice were implanted s.c. with different histological types of human and murine tumors, respectively. BMS-214662 was administered both parenterally and p.o. and was active by all these routes. Curative responses were observed in mice bearing staged human tumor xenografts including HCT-116 and HT-29 colon, MiaPaCa pancreatic, Calu-1 lung, and EJ-1 bladder carcinomas. A subline of HCT-116, HCT-116/VM46, resistant to many standard cytotoxic agents by means of a multiple drug resistance mechanism, remained quite susceptible to BMS-214662, and borderline activity was achieved against N-87 human gastric carcinoma. Two murine tumors, Lewis lung carcinoma and M5076 sarcoma, were insensitive to the FTI. In a study performed using Calu-1 tumor-bearing mice, no obvious schedule dependency of BMS-214662 was observed. The FTI, BMS-214662, demonstrated broad spectrum activity against human tumors, but murine tumors were not as sensitive.

Exposure of the human malignant peripheral nerve sheath tumor cell lines STS-26T, ST88-14, and NF90-8 to nanomolar concentrations of both lovastatin and farnesyl transferase inhibitor (FTI)-1 but not to either drug alone induced cell death. ST88-14 and NF90-8 cells underwent apoptosis, yet dying STS-26T cells did not. FTI-1 cotreatment induced a strong and sustained autophagic response as indicated by analyses of microtubule-associated protein-1 light chain 3 (LC3)-II accumulation in STS-26T cultures. Extensive colocalization of LC3-positive punctate spots was observed with both lysosome-associated membrane protein (LAMP)-1 and LAMP-2 (markers of late endosomes/lysosomes) in solvent or FTI-1 or lovastatin-treated STS-26T cultures but very little colocalization in lovastatin/FTI-1-cotreated cultures. The absence of colocalization in the cotreatment protocol correlated with loss of LAMP-2 expression. Autophagic flux studies indicated that lovastatin/FTI-1 cotreatment inhibited the completion of the autophagic program. In contrast, rapamycin induced an autophagic response that was associated with cytostasis but maintenance of viability. These studies indicate that cotreatment of STS-26T cells with lovastatin and FTI-1 induces an abortive autophagic program and nonapoptotic cell death.

Chronic myeloid leukemia (CML) typically runs a biphasic or triphasic course, with diagnoses usually made in the chronic phase (CP). Without effective treatment, patients eventually progress to a blastic phase (BP), frequently through an intermediate or accelerated phase (AP). Because the definition of AP varies among studies, comparisons of outcome and prognosis are difficult. The management of patients in these advanced phases of the disease has been much less satisfactory than that of patients in CP. Treatment with interferon-alfa (IFNalpha)-based therapy is ineffective for most patients in AP and for all of those in BP. Imatinib mesylate has demonstrated significant activity AP and BP disease, although the results are inferior compared to treatment in CP. In AP, 82% of patients achieve a hematologic response, with 24% achieving a major cytogenetic remission (MCR). Early MCR (within 3 months of diagnosis) provides a survival advantage over patients who do not achieve this response or achieve it later. In BP, 21% of previously treated patients and 36% of previously untreated patients have responded to imatinib, and up to 17% of patients may achieve a major cytogenetic response. However, responses are frequently short-lived. Several agents are being investigated for treatment of advanced-phase CML, including decitabine (DAC), homoharringtonine (HHT), troxacitabine, clofarabine, farnesyl transferase (FTase) inhibitors (FTI), and others. Many have also proven to be synergistic with imatinib in vitro and combination studies are ongoing. Continued investigation of these approaches is needed to improve the long-term prognosis of advanced-phase CML.