Resistance to cytotoxic drugs frequently emerges during treatment of leukemia with conventional chemotherapy. New classes of anticancer drugs, such as the farnesyltransferase inhibitors (FTIs), show therapeutic promise, but whether cells will easily develop resistance against them is not known. Here, we grew breakpoint cluster region/Abelson murine leukemia (Bcr/Abl) P190 lymphoblasts on stroma and made them resistant to the FTI SCH66336/lonafarnib to model emerging drug resistance in a patient. These cells exhibited greatly increased >> 100-fold) expression levels of a novel ATP (adenosine triphosphate)-binding cassette (ABC) transporter-homologous gene, ATP11A. We showed that overexpression of this gene provided protection against the effects of SCH66336, whereas knockdown of endogenous ATP11a using small interfering RNA (siRNA) made cells more sensitive to this drug. The lymphoblasts that were resistant to this FTI were also more resistant to FTI-276 and to GGTI-298, 2 other structurally similar inhibitors. Surprisingly, the cells were also able to survive higher concentrations of imatinib mesylate, the Bcr/Abl tyrosine kinase inhibitor. However, the cells remained sensitive to vincristine. Our results show that elevated levels of ATP11a can protect malignant lymphoblastic leukemia cells against several novel small molecule signal transduction inhibitors. A determination of the expression levels of this gene may have prognostic value when treatment with such classes of drugs is contemplated.

Farnesyl transferase inhibitors (FTIs) are novel antitumor drugs with clinical activity. FTIs inhibit cell growth not only by preventing direct Ras farnesylation but also through a Ras-independent pathway. Proteomics has been shown to be a powerful tool to monitor and analyze molecular networks and fluxes within the living cells and to identify the proteins that participate in these networks upon perturbation of the cellular environment. To observe early and dynamic protein changes in the cellular response to FTI in ovarian cancer cells, total proteins were extracted from 2774 cells treated or not with 10 microM manumycin, an FTI, for 3, 6 and 16 h. The proteins in the cells that were differentially expressed following treatment with manumycin for 3, 6 and 16 h were noted by two-dimensional electrophoresis and further identified by peptide mass fingerprinting as stress proteins. Both heat shock protein 70 (HSP70) and altered HSP70 were significantly up-regulated as early as 16 h in 2774 cells after exposure to manumycin. Since HSP70 plays an important role in protecting cells under stress, we treated the 2774 cells with the HSP inhibitor quercetin in combination with FTI. Quercetin dramatically enhanced the manumycin-mediated apoptosis in 2774 cells. Inducible HSP70 by manumycin in surviving ovarian cancer cells was also inhibited by quercetin as demonstrated by enzyme-linked immunosorbent assay. The inhibition of HSP70 by quercetin was correlated with enhancement of manumycin-induced mediated apoptosis in 2774 cells. The inhibition of HSP70 by 50 microM quercetin was also correlated with a decreased expression of procaspase-3 and enhancement of specific cleavage of poly (ADP-ribose) polymerase into apoptotic fragment in 2774 cells treated with manumycin. The interaction between the HSP70 inhibitor and FTI confirms the functional significance of the up-regulation of HSP70 as a protective mechanism against FTI-induced apoptosis and provides the framework for combination treatment of ovarian cancer.

Adult T-cell leukemia (ATL) is a fatal lymphoproliferative disease that develops in human T-cell leukemia virus type I (HTLV-I)-infected individuals. Despite the accumulating knowledge of the molecular biology of HTLV-I-infected cells, effective therapeutic strategies remain to be established. Recent reports showed that the hydroxyl-3-methylglutaryl (HMG)-CoA reductase inhibitor statins have anti-proliferative and apoptotic effects on certain tumor cells through inhibition of protein prenylation. Here, we report that statins hinder the survival of ATL cells and induce apoptotic cell death. Inhibition of protein geranylgeranylation is responsible for these effects, since simultaneous treatment with isoprenoid precursors, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, but not a cholesterol precursor squalene, restored the viability of ATL cells. Simvastatin inhibited geranylgeranylation of small GTPases Rab5B and Rac1 in ATL cells, and a geranylgeranyl transferase inhibitor GGTI-298 reduced ATL cell viability more efficiently than a farnesyl transferase inhibitor FTI-277. These results not only unveil an important role for protein geranylgeranylation in ATL cell survival, but also implicate therapeutic potentials of statins in the treatment of ATL.

Farnesyl transferase inhibitors (FTIs) were shown to be effective in modulating tumor growth in Ras-transformed tumor cells. Recent studies have focused on Rho GTPases as putative targets of FTI action. Previously, we demonstrated that FTIs were effective in inhibiting the growth and invasiveness of RhoC GTPase-overexpressing inflammatory breast cancer (IBC) cells however, RhoC activity was increased. In this study, we examine the mechanisms of FTI action on breast cancer cells in culture through modulation of RhoC and RhoA GTPases. We found that FTI inhibition of breast cancer cell growth was reversible and resembled what has been described for an in vitro model of tumor cell dormancy. On FTI treatment, levels of active RhoA decreased significantly, whereas levels of active RhoC increased 3.8-fold. We studied the role of these two GTPases in a fibronectin and basic FGF-induced model of breast cancer cell dormancy. Hypoactivation of RhoA and hyperactivation of RhoC were seen to induce morphology and growth changes consistent with tumor cell dormancy in culture. In addition, the JNK/SAPK pathway was induced on FTI treatment. A pharmacologic inhibitor of the JNK/SAPK pathway significantly reduced the number of dormant cells. This study has implications for the use of FTIs as therapeutic agents as well as potential mechanisms for breast cancer cell dormancy.

The combination of farnesyltransferase inhibitors (FTIs) and taxanes has been shown to result in potent antiproliferative and antimitotic synergy. Recent phase I and II clinical trials have shown that this combination shows clinical activity in taxane-refractory or taxane-resistant cancer patients. To understand the mechanism behind these clinical observations, we used a cancer cell model of paclitaxel resistance and showed that the FTI/taxane combination retains potent antiproliferative, antimitotic, and proapoptotic activity against the paclitaxel-resistant cells, at doses where each drug alone has little or no activity. To probe the mechanistic basis of these observations, paclitaxel activity was monitored in living cells using the fluorescently conjugated paclitaxel, Flutax-2. We observed that all FTIs tested increase the amount of microtubule-bound Flutax-2 and the number of microtubules labeled with Flutax-2 in both paclitaxel-resistant and paclitaxel-sensitive cells. Importantly, we observed a consequential increase in microtubule stability and tubulin acetylation with the combination of the two drugs, even in paclitaxel-resistant cells, confirming that the enhanced taxane binding in the presence of FTI affects microtubule function. Furthermore, this mechanism is dependent on the function of the tubulin deacetylase, HDAC6, because in cells overexpressing a catalytically inactive HDAC6, FTIs are incapable of enhancing Flutax-2-microtubule binding. Similar results were obtained by using an FTI devoid of farnesyltransferase inhibitory activity, indicating that functional inhibition of farnesyltransferase is also required. Overall, these studies provide a new insight into the functional relationship between HDAC6, farnesyltransferase, and microtubules, and support clinical data showing that the FTI/taxane combination is effective in taxane-refractory patients.

BACKGROUND

Catheter-tissue contact is important for effective lesion creation.

OBJECTIVE

To assess the effect of respiration on contact force (CF) during atrial fibrillation and cavotricuspid isthmus (CTI)-dependent atrial flutter ablation.

METHODS

Patients undergoing CTI ablation alone (n = 15) and pulmonary vein (PV) isolation alone (n = 12) under general anesthesia were recruited. Lesions were delivered under ventilation (30 seconds) alternating with lesions delivered under apnea (30 seconds) at an adjacent anatomical site at CTI or PV antra. The average force (F(av)), force-time integral (FTI), and force variability were measured in a region-specific manner by using a novel CF-sensing ablation catheter. Operators were blinded to CF data.

RESULTS

F(av) and FTI were higher with apnea than with ventilation in all CTI and PV segments (P <.05), an effect attributed to drop in CF with each respiratory swing, resulting in greater force variability during ventilation (P <.05). Low FTI lesions (<500 g) were strongly associated with longer ablation time to achieve bidirectional CTI block (r(2) = .81; P <.001), left PVI (r(2) = .65; P = .009), and right PVI (r(2) = .41; P = .05). Sites with transient CTI block were associated with lower F(av) and FTI than were sites with persistent CTI block (P <.05). Sites of acute PV reconnection were associated with lower F(av) and FTI compared with non-reconnected sites (P <.001).

CONCLUSIONS

Catheter-tissue CF is critically influenced by respiration; greater CF is observed with ablation during apnea. Poor CF is implicated in longer ablation time to achieve CTI block or PV isolation and in acute reconnection.

Farnesyltransferase inhibitors (FTIs) have been demonstrated to induce growth arrest or apoptosis independent of Ras mutation. Alternatively, Akt has been proposed as a potential target for the FTI's actions. This study investigated whether Lonafarnib was effective in inhibiting the growth of human nonsmall cell lung cancer (NSCLC) cells and elucidated the role of Akt in mediating such growth inhibitory effects. Lonafarnib, at clinical achievable concentration ranges, was effective in inhibiting the growth of 10 NSCLC cell lines, particularly after a prolonged treatment, regardless of Ras mutational status. Lonafarnib arrested cells growth at G(1) or G(2)/M phase in the majority tested cell lines. However it induced apoptosis when cells were cultured in a low serum (0.1%) medium. The majority of NSCLC cell lines expressed undetectable level of phosphorylated Akt (p-Akt). Lonafarnib at up to 10 muM did not decrease either total Akt level or p-Akt level in any of the tested cell lines, even after a 48 h treatment. Unexpectedly, Lonafarnib even increased p-Akt level in one cell line, although it was as sensitive as others to Lonafarnib treatment and underwent G(2)/M arrest. Bovine serum albumin completely rescued cells from Lonafarnib-induced apoptosis in low serum medium, indicating that proteins rather than cytokines or growth factors in serum masks Lonafarnib's pro-apoptotic effect. Therefore, we conclude that Lonafarnib is effective in inhibiting the growth of NSCLC cells either via growth arrest or induction of apoptosis without downregulation of Akt.

In previous studies we found that the GTPase p21 Harvey-Ras (Ha-Ras) stimulates the production of reactive oxygen species and induces apoptosis by oxidative stress; this effect was reversed by farnesyl transferase inhibitors (FTIs). In this study we investigated whether FTIs reduce rat brain damage induced by an excitotoxic stimulus, and the signalling pathway(s) underlying the neuroprotection by FTIs. In brain tissue, protein levels of Ha-Ras and farnesylation inhibition were assayed by Western blot, and superoxide production was measured by hydroethidine. The excitotoxic lesion was induced by intrastriatal injection of N-methyl-d-aspartate (NMDA). The survival of mouse neuronal cortical cells was assessed by 3-(4,5 dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In brain tissue, NMDA increased the protein levels of Ha-Ras, FTIs caused the accumulation of non-prenylated inactive Ras in the cytosolic fraction, and significantly reduced superoxide production and necrotic volume after excitotoxicity. FTIs increased the viability of mouse neuronal cortical cells following oxidative stress. In conclusion, FTIs inhibited Ha-Ras, decreased oxidative stress and reduced necrotic volume by partly acting on neuronal cells. Thus, Ha-Ras inhibition plays a role in the pathology of neuroprotection, suggesting a potential role of FTIs in the treatment of cerebrovascular diseases.

Farnesyltransferase inhibitors (FTIs) inhibit certain cellular signal transduction pathways, and are being evaluated for activity in hematologic malignancies. Tipifarnib and lonafarnib are orally available FTIs that are active against a variety of targets and inhibit several pathways involved in the pathogenesis of hematologic malignancies. FTIs have demonstrated activity in a variety of hematologic diseases, including acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, and multiple myeloma. This article reviews the clinical experience with tipifarnib and lonafarnib in the treatment of hematologic malignancies.

OBJECTIVE

Assessment of disease severity for patients with hyperthyroidism involves clinical evaluation and laboratory testing. To determine if there is a correlation between symptoms and thyroid function test results, we prospectively studied hyperthyroid patients using a standardized symptom rating scale and serum thyroid function parameters.

METHODS

We examined 25 patients with untreated, newly diagnosed Graves' disease using the Hyperthyroid Symptom Scale (HSS) and serum levels of thyroxine (T4), triiodothyronine (T3) relative insulin area (RIA), and estimates of free thyroxine index (FTI). In addition, we compared thyroid hormone levels with standard measures of depression and anxiety in these patients.

RESULTS

When regression analyses controlling for age were performed, none of these symptom ratings were associated with FTI or T3 RIA. The HSS was correlated with goiter size and anxiety ratings and was inversely correlated with age.

CONCLUSIONS

The present study suggests that there is no relationship between the clinical assessment of disease severity and serum levels of thyroid hormone in untreated Graves' disease.

The c-myb oncogene is a frequent target for retroviral activation in hemopoietic tumors of avian and mammalian species. While insertions can target the gene directly, numerous clusters of retroviral insertion sites have been identified which map close to c-myb and outside the transcription unit in T-lymphomas (Ahi-1, fit-1, and Mis-2) and monocytic and myeloid leukemias (Mml1, Mml2, Mml3, and Epi-1). Previous analyses showed no consistent effect of these insertions on c-myb expression, raising the possibility that other nearby genes were the true targets. In contrast, our analysis of four cell lines established from lymphomas bearing insertions at fit-1 (fti-1) (feline leukemia virus) and Ahi-1 (Moloney murine leukemia virus) shows that these display higher expression levels of c-myb RNA and protein compared to a panel of phenotypically similar cell lines lacking such insertions. An interesting feature of the cell lines with long-range c-myb insertions was that each also carried an activated Myc allele. The potential for oncogenic synergy between Myb and Myc in T-cell lymphoma was confirmed in transgenic mice overexpressing alleles of both genes in the T-cell compartment, lending further credence to the case for c-myb as the major target for long-range activation. In contrast, mapping and analysis of c-myb neighboring genes (HBS1 and FLJ20069) showed that the expression of these genes did not correlate well with the presence of proviral insertions. A possible explanation for the paradoxical behavior of c-myb was provided by one of the murine T-lymphoma lines bearing an insertion at Ahi-1 (p/m16i) that reproducibly down-regulated c-myb RNA and protein to very low levels or undetectable levels on prolonged culture. Our observations implicate c-myb as a key target of upstream and downstream retroviral insertions. However, overexpression may become dispensable during outgrowth in vitro, and perhaps during tumor progression in vivo, providing a potential rationale for the previously observed discordance between retroviral insertion and c-myb expression levels.

Local recurrence after a definitive course of radiation therapy remains a significant clinical problem and represents a common pattern of failure for many solid tumors. The sensitivity of tumor cells to the cytotoxicity of ionizing radiation is thought to be one of the major determinants of local control for tumors in patients treated with radiation therapy. There is substantial experimental evidence to demonstrate that increased radiation resistance is associated with the expression of activated oncogenes, including Ras. Mutated forms of Ras are found in 30% of human cancers including a substantial proportion of pancreatic and colon adenocarcinomas. Mutated Ras produces proteins that remain locked in a constitutively active state, thereby relaying uncontrolled signals. Ras proteins are guanosine triphosphate-binding proteins that play a pivotal role in the control of many cellular processes, including growth and differentiation. Preclinical studies have shown that expression of mutant Ras increases cellular radioresistance. Ras function is dependent on its localization to the plasma membrane. This is achieved by posttranslational modifications, including the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (FTase). This enzyme has become an important target for the design of new agents that target Ras. FTase inhibitors (FTIs) block the farnesylation of Ras and reverse Ras-mediated radioresistance in human cell lines. FTIs have been well tolerated in animal studies and appear not to cause generalized cytotoxicity. There are ongoing clinical trials to determine the optimal therapeutic schedules and dose for FTIs. A phase 1 trial of the FTI L778-123 and radiotherapy has recently been completed.

OBJECTIVE

Preliminary studies have demonstrated that the Ras family and related guanosine 5'-triphosphate-dependent proteins (G-proteins) are overactivated in malignant gliomas and may function as indirect mediators of glial transformation initiated by deregulated upstream signaling elements. We postulated that inhibiting the activation of such proteins might represent a promising strategy for blocking the aberrant proliferation of these tumors.

RESULTS

Accordingly, we examined the therapeutic efficacy against malignant glioma cells in vitro of a series of selective peptidomimetic inhibitors of farnesylation (FTI-277) and geranylgeranylation (GGTI-286 and GGTI-298), which are critical steps in the post-translational processing (prenylation) of these proteins. We first defined concentration-response relationships for each of these agents, using MTS-based cell proliferation assays in the established malignant glioma cell lines U-87 and LN-Z308 and the low-passage malignant glioma cell line SG-388. FTI-277, GGTI-286, and GGTI-298 each produced a striking concentration-dependent antiproliferative effect on the glioma cell lines, with the median effective dose ranging from 2.5 to 15.5 micromol/L. We then assessed the effect of prenylation inhibition on cell viability using clonogenic growth assays. This demonstrated a steady drop in the number of colonies with increasing drug concentrations for all three inhibitors. Third, we examined whether the cytotoxic effects of one of these inhibitors (GGTI-298) were associated with the induction of apoptosis using a terminal transferase-catalyzed in situ end-labeling technique. This approach showed a time-dependent increase in apoptotic cell numbers, which correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion.

CONCLUSIONS

Our studies demonstrated that FTI-277, GGTI-286, and GGTI-298 each yielded significant antiproliferative effects in human malignant glioma cells in vitro at low micromolar concentrations, which have been achievable in vivo without major systemic toxicity. Extended periods of drug treatment produced cytotoxicity in the tumor cells, which correlated with the induction of apoptosis. We conclude that inhibition of Ras and related G-proteins offers a promising approach for blocking glioma proliferation that justifies further investigation in vivo.

Both systolic pressure-volume area (PVA) and force-time integral (FTI) have been used as measures of oxygen consumption per beat (VO2) in the isolated left ventricle. The reciprocal of the slope of the VO2-PVA relation has been considered to reflect the chemomechanical energy transduction efficiency of the contractile machinery (contractile efficiency), whereas its VO2 intercept consists of energy cost of excitation-contraction coupling and basal metabolism. To examine whether the increase in myosin isoform V1/V3 ratio in hyperthyroid rabbits decreases contractile efficiency and to determine overall mechanisms of higher oxygen consumption in hyperthyroid hearts, the VO2-PVA and VO2-FTI relations as well as the end-systolic pressure-volume relation were assessed in cross-circulated, isovolumically beating hearts isolated from normal, hyperthyroid, and hypothyroid rabbits. Normalized initial slopes of the rising limb of the curvilinearly fitted end-systolic pressure-volume relation (E'max, ventricular contractility index) were similar for normal and hyperthyroid groups. However, the slopes and VO2 intercepts of the VO2-PVA and VO2-FTI relations were greater in hyperthyroid hearts than in normal hearts. Accordingly, in the hyperthyroid hearts, the contractile efficiency (27 +/- 6%) was lower and left ventricular VO2 for excitation-contraction coupling (0.028 +/- 0.004 ml O2/beat/100 g) was higher than in normal hearts (40 +/- 4% and 0.021 +/- 0.005 ml O2/beat/100 g, respectively). This decreased contractile efficiency in the hyperthyroid hearts was attributable to myosin isoform alteration rather than to increased beta-adrenoceptors because isoproterenol did not affect the slope of the VO2-PVA relation in all groups. In contrast, the slope of the VO2-FTI relation was significantly increased by isoproterenol in all groups. Neither the VO2-PVA nor the VO2-FTI relations in hypothyroid hearts were different from those in normal hearts except for significantly lower VO2 for basal metabolism. We conclude that in hyperthyroid rabbits, the left ventricle has decreased contractile efficiency and increased energy cost of excitation-contraction coupling and that the decreased contractile efficiency in hyperthyroid hearts is probably due to the increased V1/V3 ratio of the myosin isoform component. In addition, this study demonstrates that the VO2-PVA and VO2-FTI relations dissociate depending on the intervention, even in the same isovolumic contraction mode.

BACKGROUND

This paper presents a nationwide analysis of suicide mortality in Finland from 1990 to 1995, when the total use of antidepressants, especially that by selective serotonin reuptake inhibitors (SSRIs) expanded in the country.

METHODS

Suicide rate was analysed by various methods including that by intake of antidepressants. Various antidepressants were compared by calculating fatal toxicity indices (FTI) by relating number of fatal poisonings by a drug to its consumption.

RESULTS

The expanded use of antidepressants coincided with an increased number of deaths caused by these drugs. The proportion of suicides committed by use of antidepressants among all suicides increased from 5.6% to 8.4%. The total suicide rate, however, declined significantly. This was mainly accounted for by the reduced suicide rates by hanging and carbomonoxide poisoning, which outnumbered the increased figures of suicides by poisoning. On the whole, 82% of suicides by antidepressants were committed by use of tricyclics. Use of doxepin and amitriptyline remained steady, and their FTIs were constantly high. The lowest FTIs were associated with fluoxetine, citalopram, mianserin and moclobemide.

CONCLUSIONS

The method ignores causality between the increased use of SSRIs and suicide mortality. Various factors affecting risk of suicide or choice of a method remain outside the scope of the data.

CONCLUSIONS

The increased use of SSRIs coincided with a significant decline in suicide mortality. However, suicides by use of antidepressants showed an upward trend. Therefore, in suicide prevention, risks and benefits of antidepressants should be considered in choosing treatment for depressive patients.

High amounts of nitric oxide (NO) produced by activated macrophages or NO donors are required to induce cytotoxicity and apoptosis in pathogens and tumor cells. High concentrations of NO may lead to nonspecific toxicity thereby limiting the use of NO donors in the treatment of cancer. In this study, we tested the possibility of potentiating the apoptotic action of NO in a human breast cancer cell line, MDA-MB-468, by combining it with a farnesyltransferase inhibitor (FTI), which has been shown to induce apoptosis in some other cancer cell lines with minimal toxicity to normal cells. DETA-NONOate, a long acting NO donor which has a half-life of 20 h at 37 degrees C, was used in this study. DETA-NONOate (1 mM), which releases NO in the range produced by activated macrophages, induced apoptosis after 36 h in MDA-MB-468 cells via cytochrome c release and caspase-9 and -3 activation. FTI (25 microM) potentiated the action of lower concentrations of DETA-NONOate (25-100 microM) by inducing apoptosis in these cells within 24 h by increasing cytochrome c release and caspase-9 and -3 activation. This effect was observed preferentially in the cancer cell lines studied with no apoptosis induction in normal breast epithelial cells. This novel combination of FTI and NO may emerge as a promising approach for the treatment of breast cancer.

Farnesyltransferase inhibitors (FTIs) usually cause growth inhibition, but in certain preclinical settings they have been shown to induce apoptosis, a clinically desirable response. In this study, we show that the proapoptotic effects of FTIs in Ras-transformed cells are masked by activation of phosphatidylinositol 3'-kinase (PI3'K) or AKT, which are controlled by cytokines and integrins. The results implied that FTIs disrupt a signal that is crucial for survival of malignant cells, but not normal cells, if the PI3'K-AKT pathway is inactivated. Our findings have implications for clinical applications of FTIs where apoptotic responses would be preferred.

We identified dexamethasone-induced Ras protein 1 (Dexras1) as a negative regulator of protein kinase C (PKC) delta, and the consequences of this regulation have been examined for adenylyl cyclase (EC 4.6.1.1) type 2 (AC2) signaling. Dexras1 expression in human embryonic kidney 293 cells completely abolished dopamine D2 receptor-mediated potentiation of AC2 activity, which is consistent with previous reports of its ability to block receptor-mediated Gbetagamma signaling pathways. In addition, Dexras1 significantly reduced phorbol 12-myristate 13-acetate (PMA)-stimulated AC2 activity but did not alter Galpha(s)-mediated cAMP accumulation. Dexras1 seemed to inhibit PMA stimulation of AC2 by interfering with PKCdelta autophosphorylation. This effect was selective for the delta isoform because Dexras1 did not alter autophosphorylation of PKCalpha or PKCepsilon. Dexras1 disruption of PKCdelta autophosphorylation resulted in a significant blockade of PKC kinase activity as measured by [gamma-32P]ATP incorporation using a PKC-specific substrate. Moreover, Dexras1 and PKCdelta coimmunoprecipitated from whole-cell lysates. Dexras1 did not alter the membrane translocation of PKCdelta; however, the ability of Dexras1 to interfere with PKCdelta autophosphorylation was isoprenylation-dependent as determined using the farnesyltransferase inhibitor methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and a CAAX box-deficient Dexras1 (C277S) mutant. PMA-stimulated AC2 activity was also not affected by Dexras1 C277S. Taken as a whole, these data suggest that Dexras1 functionally interacts with PKCdelta at the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKCdelta activity. Moreover our study suggests that Dexras1 acts to modulate the activation of AC2 in an indirect fashion by inhibiting both Gbetagamma- and PKC-stimulated AC2 activity. The current study provides a novel role for Dexras1 in signal transduction.

Diastolic dysfunction at high heart rates may be associated with increased myocardial energy consumption. Frequency-dependent changes of isometric force and oxygen consumption (MVO2) were investigated in strip preparations from endstage failing human hearts exhibiting various degrees of diastolic dysfunction. MVO2 was determined by a new method which was validated. When stimulation rate was increased from 40 to 200 min-1 (n=7), developed force decreased from 16.5+/-4.3 to 7.9+/-2.9 mN/mm2 (P<0.01), diastolic force increased from 15.9+/-3.2 to 22.0+/-3.0 mN/mm2 (P<0.01), and total MVO2 increased from 2.6+/-0.6 to 4.7+/-0.9 ml/min/100 g (P<0.025). Resting MVO2 and resting force were 1.8+/-0.4 ml/min/100 g and 15.9+/-3.0 mN/mm2, respectively. After addition of 30 mm 2,3-butanedione monoxime (BDM) to inhibit crossbridges, resting MVO2 and resting force decreased by 46% (P<0.05) and 15% (P<0.01), respectively, indicating the presence of active force generation in unstimulated failing human myocardium. In each muscle preparation, there was a significant correlation between force-time integral (FTI) and total MVO2 (r=0.96+/-0.01). The strength of these correlations did not vary with the contribution of diastolic FTI to total FTI. The ratio of activity related MVO2 to developed FTI, an inverse index of the economy of contraction, increased depending on the rise of diastolic FTI at higher stimulation rates. In conclusion, in failing human myocardium, diastolic force development is occurring at the same energy expenditure as systolic force generation. Therefore, in muscle preparations with disturbed diastolic function economy of contraction decreases with higher stimulation rates, depending on the rise of diastolic force.

Activated ras is known to dysregulate TGF-beta signaling by altering the expression of TGF-beta type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-beta signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 microM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 microM concentrations (P>0.004). The TGF-beta effector gene p21(waf1/cip1) was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-beta responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-beta signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT-PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-beta signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

BACKGROUND

Enormous progress has been made in the past 5 years in our understanding of the gene products governing the response of mammalian cells to ionizing radiation. Many of these are potential targets for enhancing the effectiveness of radiotherapy. However, a major barrier to such efforts is the requirement for a preferential effect on tumor vs. normal cells. Such a requirement can only come about by exploiting a known difference between tumor and normal cells.

METHODS

This review highlights three differences between tumor and normal cells that are being exploited with fractionated radiotherapy.

RESULTS

The three strategies to enhance preferentially tumor response to radiotherapy are inhibition of ras activity using farnesyltransferase inhibitors (FTIs), inhibition of epidermal growth factor receptors (EGFRs), and the use of drugs that preferentially kill hypoxic cells. Each of these strategies exploits a known difference between at least some tumors and their surrounding normal tissues, and each has shown encouraging results when combined with fractionated radiation in preclinical studies.

CONCLUSIONS

For each of the three strategies to enhance preferentially the sensitivity of cancers, the preclinical and early clinical data are promising for their successful application in radiotherapy.

Recently, we have shown that the farnesyltransferase inhibitor FTI-2153 induces accumulation of two human lung cancer cell lines in mitosis by inhibiting bipolar spindle formation during prometaphase. Here we investigate whether this mitotic arrest depends on transformation, Ras and/or p53 mutation status. Using DAPI staining (DNA) and immunocytochemistry (microtubules), we demonstrate that in normal primary foreskin fibroblasts (HFF), as well as in several cancer cell lines of different origins including human ovarian (OVCAR3), lung (A-549 and Calu-1) and fibrosarcoma (HT1080), FTI-2153 inhibits bipolar spindle formation and induces a rosette morphology with a monopolar spindle surrounded by chromosomes. In both malignant cancer cell lines and normal primary fibroblasts, the percentage of prometaphase cells with bipolar spindles decreases from 67-92% in control cells to 2-28% in FTI-2153 treated cells. This inhibition of bipolar spindle formation correlates with an accumulation of cells in prometaphase. The ability of FTI-2153 to inhibit bipolar spindle formation is not dependent on p53 mutation status since both wild-type (HFF, HT1080 and A-549) and mutant (Calu-1 and OVCAR3) p53 cells were equally affected. Similarly, both wild-type (HFF and OVCAR3) and mutant (HT1080, Calu-1 and A-549) Ras cells accumulate monopolar spindles following treatment with FTI-2153. However, two cell lines, NIH3T3 (WT Ras and WT p53) and the human bladder cancer cell line, T-24 (mutant H-Ras and mutant p53) are highly resistant to FTI-2153 inhibition of bipolar spindle formation. Finally, the ability of FTI-2153 to inhibit tumor cell proliferation does not correlate with inhibition of bipolar spindle formation. Taken together these results demonstrate that the ability of FTI-2153 to inhibit bipolar spindle formation and accumulate cells in mitosis is not dependent on transformation, Ras or p53 mutation status. Furthermore, in some cell lines, FTIs inhibit growth by mechanisms other than interfering with the prophase/metaphase traverse.

Protein farnesyl transferase (FTase) catalyzes transfer of a 15-carbon farnesyl group from farnesyl diphosphate (FPP) to a conserved cysteine in the C-terminal Ca1a2X motif of a range of proteins ("C" refers to the cysteine, "a" to any aliphatic amino acid, and "X" to any amino acid), and the lipid chain interacts with, and forms part of, the Ca1a2X peptide binding site. Here, we employed a library of anilinogeranyl diphosphate (AGPP) derivatives to examine whether altering the interacting surface between the two substrates could be exploited to generate Ca1a2X peptide selective FPP analogues. Analysis of transfer kinetics to dansyl-GCVLS peptide revealed that AGPP analogues with substituents smaller than or equal in size to a thiomethyl group supported FTase function, while analogues with larger substituents did not. Analogues with small meta-substitutions on the aniline ring such as iodo and cyano increased reactivity with dansyl-GCVLS and provided analogues that were effective FPP competitors. Other analogues with ortho-substitutions on the aniline were potent dansyl-GCVLS modification FTase inhibitors (Ki in the 2.4-18 nM range). Both meta- and para-trifluoromethoxy-AGPP are transferred to dansyl-GCVLS while the ortho-substituted isomer was a potent farnesyl transferase inhibitor (FTI) with an inhibition constant Ki = 3.0 nM. In contrast, ortho-trifluoromethoxy-AGPP was efficiently transferred to dansyl-GCVIM. Competition for dansyl-GCVLS and dansyl-GCVIM peptides by FPP and ortho-trifluoromethoxy-AGPP gave both analogue and farnesyl modified dansyl-GCVIM but only farnesylated dansyl-GCVLS. We provide evidence that competitive modification of dansyl-GCVIM by ortho-trifluoromethoxy-AGPP stems from a prechemical step discrimination between the competing peptides by the FTase-analogue complex. These results show that subtle changes engineered into the isoprenoid structure can alter the reactivity and FPP competitiveness of the analogues, which may be important for the development of prenylated protein function inhibitors.

There is an increasing rationale for effective combinations of endocrine therapy with novel drugs that target aberrant signal transduction pathways in estrogen receptor (ER) positive breast cancer. Prolonged endocrine therapy can be associated with an acquired increase in peptide growth factor signaling (EGFR, HER2), together with cross-talk activation of ER-dependent gene transcription and cell growth that leads to endocrine resistance. Current approaches to target these pathways include both the selective ER downregulator fulvestrant, and various signal transduction inhibitors (STIs). Fulvestrant can overcome resistance to tamoxifen (TAM-R) and long-term estrogen deprivation (LTED-R) in experimental models by reducing ER expression, and represents a current option for post-menopausal women with endocrine resistant ER+ve breast cancer. Emerging data suggest that fulvestrant's effect may be greater when combined with estrogen deprivation, and several phase III trials are assessing fulvestrant combined with aromatase inhibitors (AIs). Small molecule STIs such as tyrosine kinase inhibitors (TKIs), farnesyltransferase inhibitors (FTIs) and mTOR antagonists are also active in breast cancer. Pre-clinical data suggest that combined endocrine/STI therapy may result in greater growth inhibition than either therapy alone, and thus delay emergence of resistance. Several clinical trials are now examining STIs combined with AIs both in the tamoxifen-resistant and first-line advanced breast cancer setting, while pre-surgical studies are investigating the efficacy of combined endocrine/STI therapy utilising biological primary endpoints. This article reviews the pre-clinical rationale for this strategy and the clinical trials in this area.