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PDF file, 1202KB, Supplementary Figure 1. Kaplan-Meier Survival Curves. Supplementary Figure 2. Kaplan-Meier Survival Curves. Supplementary Table 1. Univariate analysis of clinical covariates.

Purpose: This study assesses the ability of multidrug resistance (MDR)–associated gene expression patterns to predict survival in patients with newly diagnosed carcinoma of the ovary. The scope of this research differs substantially from that of previous reports, as a very large set of genes was evaluated whose expression has been shown to affect response to chemotherapy.Experimental Design: We applied a customized TaqMan low density array, a highly sensitive and specific assay, to study the expression profiles of 380 MDR-linked genes in 80 tumor specimens collected at initial surgery to debulk primary serous carcinoma. The RNA expression profiles of these drug resistance genes were correlated with clinical outcomes.Results: Leave-one-out cross-validation was used to estimate the ability of MDR gene expression to predict survival. Although gene expression alone does not predict overall survival (OS; P = 0.06), four covariates (age, stage, CA125 level, and surgical debulking) do (P = 0.03). When gene expression was added to the covariates, we found an 11-gene signature that provides a major improvement in OS prediction (log-rank statistic P < 0.003). The predictive power of this 11-gene signature was confirmed by dividing high- and low-risk patient groups, as defined by their clinical covariates, into four specific risk groups on the basis of expression levels.Conclusion: This study reveals an 11-gene signature that allows a more precise prognosis for patients with serous cancer of the ovary treated with carboplatin- and paclitaxel-based therapy. These 11 new targets offer opportunities for new therapies to improve clinical outcome in ovarian cancer. Clin Cancer Res; 18(11); 3197–206. ©2012 AACR.

Purpose: This study assesses the ability of multidrug resistance (MDR)–associated gene expression patterns to predict survival in patients with newly diagnosed carcinoma of the ovary. The scope of this research differs substantially from that of previous reports, as a very large set of genes was evaluated whose expression has been shown to affect response to chemotherapy. Experimental Design: We applied a customized TaqMan low density array, a highly sensitive and specific assay, to study the expression profiles of 380 MDR-linked genes in 80 tumor specimens collected at initial surgery to debulk primary serous carcinoma. The RNA expression profiles of these drug resistance genes were correlated with clinical outcomes. Results: Leave-one-out cross-validation was used to estimate the ability of MDR gene expression to predict survival. Although gene expression alone does not predict overall survival (OS; P = 0.06), four covariates (age, stage, CA125 level, and surgical debulking) do (P = 0.03). When gene expression was added to the covariates, we found an 11-gene signature that provides a major improvement in OS prediction (log-rank statistic P < 0.003). The predictive power of this 11-gene signature was confirmed by dividing high- and low-risk patient groups, as defined by their clinical covariates, into four specific risk groups on the basis of expression levels. Conclusion: This study reveals an 11-gene signature that allows a more precise prognosis for patients with serous cancer of the ovary treated with carboplatin- and paclitaxel-based therapy. These 11 new targets offer opportunities for new therapies to improve clinical outcome in ovarian cancer. Clin Cancer Res; 18(11); 3197–206. ©2012 AACR.

Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has been uncertain. Using a state-of-the-art Taqman-based quantitative RT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in established cancer cell lines (grown in monolayer, 3D scaffold, or in xenograft) and clinical samples, either containing >75% tumor cells or microdissected. The MDR transcriptome was determined a priori based on an extensive curation of the literature published during the last three decades, which led to the enumeration of 380 genes. No correlation was found between clinical samples and established cancer cell lines. As expected, we found up-regulation of genes that would facilitate survival across all cultured cancer cell lines evaluated. More troubling, however, were data showing that all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model. Although cultured cells can be used to study many aspects of cancer biology and response of cells to drugs, this study emphasizes the necessity for new in vitro cancer models and the use of primary tumor models in which gene expression can be manipulated and small molecules tested in a setting that more closely mimics the in vivo cancer microenvironment so as to avoid radical changes in gene expression profiles brought on by extended periods of cell culture.

Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and β are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.

Chemotherapy resistance in ovarian cancer remains an unsolved problem in caring for women with this disease. We now show that ovarian cancer immunoreactive antigen domain containing 1 (OCIAD1) has higher expression in chemoresistant compared with chemosensitive ovarian cancer cell lines. We have designed a novel secondary cell homing assay (SCHA) to test the ability of cells to withstand chemotherapy and form secondary colonies that could form recurrent disease. OCIAD1 upregulated cells had significantly higher secondary colony-forming ability than had OCIAD1 downregulated cells following treatment with paclitaxel. Additionally, 18:1 lysophosphatidic acid (LPA) increases OCIAD1 expression in a time- and dose-dependent manner. LPA stimulates OCIAD1 serine phosphorylation within two hours of stimulation. Transfection of MKK6 increases OCIAD1 expression but nuclear translocation is inhibited. Inhibition of p38 mitogen-activated protein kinase blocks LPA-induced OCIAD1 expression. Cycloheximide treatment of MKK6-transfected cells does not inhibit OCIAD1 expression, suggesting that MKK6 upregulation is not translationally controlled. OCIAD1 downregulation knocks down LPA-induced cell adhesion to collagen I and laminin 10/11 and specifically inhibits cell attachment to α2, α5, αV, and β1 integrins. Proteomic studies indicate that OCIAD1 is physically attached to α actin 4 and β actin. Thus, OCIAD1 may play a role in cytoskeletal function which can alter sensitivity to paclitaxel. This is the first study to indicate that OCIAD1 is a key player in generating ovarian cancer recurrence; it is functionally controlled by LPA and MKK6 signaling, and inhibition of OCIAD1 could be an important strategy in the management of recurrent ovarian cancer. Mol Cancer Ther; 9(6); 1709–18. ©2010 AACR.

We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.

Renal cell carcinoma (RCC) is recognized as a neoplasm resistant to chemotherapy. In vitro experiments demonstrated that suramin, at noncytotoxic doses, enhanced the activity of chemotherapy including 5-fluorouracil (5-FU) in xenograft models.A phase I/II trial of noncytotoxic suramin in combination with weekly 5-FU in patients with metastatic RCC was conducted. The treatment consisted of intravenous (i.v.) suramin followed by a 500 mg/m2 i.v. bolus of 5-FU given 4.5 hours after starting suramin. In the phase I portion, a cohort of 6 patients received a suramin dose calculated to achieve a plasma level of 10-50 micromol/L. Therapy was administered once weekly for 6 doses, followed by 2 weeks off. This was followed by a phase II portion in which the primary goal was to determine the objective response rate.Twenty-three patients were enrolled in the study: 6 in the phase I portion and 17 in phase II. Seventy-eight percent of patients were men, the mean age was 58.8 years, 96% had previous nephrectomy, and 70% had received previous systemic therapy. Histologic subtype was clear cell in 91%. Dose-limiting toxicity was observed in 1 of 6 patients (grade 3 hypersensitivity related to suramin infusion). The suramin dosing nomogram used in phase I and II portions of the trial yielded the desired plasma level of 10-50 micromol/L from 4.5 hours to 48 hours after infusion in 94 of 115 treatments. No objective responses were noted, and the median time to treatment failure was 2.5 months. The major toxicities (all grades) were fatigue (83%), nausea/vomiting (78%), diarrhea (61%), and chills (61%).Suramin levels expected to reverse fibroblast growth factor-induced resistance can be achieved with the dosing regimen used in this study. The toxicity observed with suramin and 5-FU was acceptable. The combination does not have clinical activity in patients with metastatic RCC.

Objectives To identify proteins unique to metastatic ovarian cancer and test their potential involvement in cell adhesion. Methods We purified plasma membrane from paired metastatic and primary tumor tissues from patients with stage IIIC ovarian cancer. Membrane proteins unique to metastases were identified by liquid chromatographic mass spectrometry (LC-MS). The role of one of the identified proteins, ovarian cancer immuno-reactive antigen domain containing 1 (OCIAD1) in cell adhesion was determined in the presence of LPA using both over-expression and down regulation approaches. Results We identified a differentially expressed 29 kDa protein as OCIAD1 over-expressed in metastatic tissues, when compared to primary tumor tissues. OCIAD1 over-expression in HEY ovarian cancer cells increased LPA-induced, but not basal level cell adhesion to extracellular matrix proteins collagen I and laminin 10/11. This enhancement was not blocked by LY294002 and GF109203X, suggesting that OCIAD1 does not use PKC and PI3K signaling pathways to exert its effect on adhesion. In addition, LPA induced cell adhesion to collagen I was unaffected by paclitaxel (5 µM) in OCIAD1 overexpressing cells. Conclusions This is the first report that OCIAD1 is over-expressed in metastatic ovarian cancer tissues. The effect of OCIAD1 on cell adhesion may be related to its function in ovarian cancer. Failure of paclitaxel to affect ovarian cancer cell adhesion in presence of OCIAD1 raises the possibility of OCIAD1's role in tumor metastasis. Ongoing studies using a mouse orthotopic LPA-dependent ovarian cancer metastasis model are focused on strategies to inhibit the potential role of OCIAD1 in tumor metastasis.

Anvirzel is an aqueous extract of the plant Nerium oleander which has been utilized to treat patients with advanced malignancies. The current study reports a phase 1 trial to determine the maximum tolerated dose (MTD) and safety of Anvirzel in patients with advanced, refractory solid tumors. Patients were randomized to receive this agent by intramuscular injection at doses of 0.1, 0.2, 0.4 ml/m2/day with subsequent patients receiving 0.8 or 1.2 ml/m2/day sequentially. Eighteen patients were enrolled and completed at least one treatment cycle of three weeks. Most patients developed mild injection site pain (78%). Other toxicities included fatigue, nausea, and dyspnea. Traditional dose limiting toxicity was not seen, but the MTD was defined by injection volume as 0.8 ml/m2/day. No objective anti-tumor responses were seen. Anvirzel can be safely administered at doses up to 1.2 ml/m2/day, with the amount administered intramuscularly limited by volume. The recommended phase II dose level is 0.8 ml/m2/day.

A simple, rapid, and sensitive analytical method for the measurement of docetaxel in human plasma was developed and validated. The method is based on positive electrospray ionization tandem mass spectrometry (ESI + –MS–MS) with on-line sample extraction. It uses paclitaxel as internal standard for calibration. The on-line sample extraction minimizes sample handling and is readily adopted for automation. Quantitation of plasma docetaxel was done by the multiple reaction monitoring (MRM) mode. The method had a linear calibration range of 1.00–3000 ng/mL with a correlation coefficient >0.9999. The limit of quantitation (LOQ) for docetaxel in plasma was 1.00 ng/mL. The on-line extraction recovery of docetaxel was between 86.1–94.7%, with %CV ≤ 6.1%. This method has high accuracy (90.1–96.3%), and excellent intra-assay (0.6–3.8%) and inter-assay (2.0–5.7%) precision. Its applicability to clinical samples was demonstrated by measuring patient plasma samples after treatment of weekly docetaxel at 25 mg/m 2 as 60-min infusion.

BACKGROUND Telomerase is a ribonucleoprotein polymerase that adds telomeric repeats to chromosome ends. This enzyme is deficient in the majority of normal somatic cells, but often is reactivated during tumorigenesis. In the current study, the authors examined telomerase activity in human American Joint Committee on Cancer Stage II colorectal carcinomas and correlated it with traditional prognostic indicators and disease outcome. METHODS The telomerase repeat amplification protocol (TRAP) was employed to determine telomerase activity in 122 surgical specimens (from 77 male and 45 female patients) of human Stage II colorectal carcinoma. The primary site of the tumor was the colon in 52 cases and the rectum in 70 cases. Telomerase activity was correlated with traditional prognostic indicators such as gender, age, T classification, tumor size, tumor grade, and disease outcome (overall survival and disease-free survival). The Median follow-up for patients who still were alive was 5.8 years. RESULTS Telomerase activity was detected in 80% of the tumors (98 of 122 tumors). Telomerase-positive patients differed from telomerase-negative patients in that they tended to be female (41% vs. 21%; P = 0.1), presented with primary tumors of the colon more frequently (49% vs. 17%; P = 0.01), and had a higher T classification (T4) (62% vs. 38%; P = 0.04). Univariate and multivariate analyses demonstrated a correlation between telomerase activity and disease-free survival (P = 0.05). CONCLUSIONS Although a large percentage of Stage II colorectal carcinoma samples were positive for telomerase activity, the prognosis for patients with telomerase-negative tumors was found to be worse than that for patients with telomerase-positive tumors. Cancer 2002;95:1834–9. © 2002 American Cancer Society. DOI 10.1002/cncr.10911

Activation of signaling pathways following DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. NF-κB, a major regulator of the stress response and a negative regulator of apoptosis is often activated following treatment with topoisomerase poisons. Since activation of NF-κB is generally considered to relay an anti-apoptotic signal, inactivation of this signaling molecule is considered to represent an important strategy to improve therapeutic efficacy. Although this strategy seems to be effective in some model systems, our results in human nonsmall cell lung cancers differed. In this review we will discuss the role of NF-κB in mediating topoisomerase poison-induced DNA damage and apoptosis and the consequence of inhibiting its activity. Newer insights about the importance of proteasome inhibitors and anti-apoptotic genes in topoisomerase poison-induced signaling mechanisms leading to apoptosis will also be reviewed. The knowledge obtained from these studi es may be useful for translation to a clinical setting for development of more effective therapeutic strategies.

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIbeta to DNA and stimulated topoisomerase IIbeta-mediated DNA cleavage at specific sites on (32)P-end-labeled DNA. Human topoisomerase IIalpha-mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chaotropic protein denaturant in the assays and selectivity for the beta-isozyme of human topoisomerase II. Studies with two topoisomerase IIbeta-negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta-isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.

Topoisomerase IIbeta knockout mouse cells (beta-/-) were found to have only slight resistance to m-AMSA, a dual topoisomerase IIalpha-IIbeta poison, as compared to wild-type cells (beta+/+) during 1 h or 3 day exposures to the drug. In contrast, the beta-/- cells were greater than threefold resistant to XK469, a selective topoisomerase IIbeta poison during three day drug exposures (beta+/+ IC(50) = 175 microM, beta-/- IC(50) = 581 microM). Short term (1 h) exposure to XK469 was not cytotoxic to either beta-/- or beta+/+ cells, suggesting that anticancer therapy with XK469 may be more efficacious if systemic levels can be prolonged. During studies on topoisomerase activity in nuclear extracts of the beta+/+ and beta-/- cells, we found evidence that XK469 is a weak topoisomerase I catalytic inhibitor. The high IC(50) for topoisomerase I inhibition (2 mM) suggests that topoisomerase I is not a significant target for XK469 cytotoxicity.

Topoisomerase II (TopoII) is a Mg-dependent enzyme involved in topological modifications of DNA that are crucial to the regulation of cell proliferation and possibly differentiation. To investigate the role of Mg availability in the modulation of TopoII in whole cells, we studied enzyme activity and expression in HL-60 cells grown in the presence of decreasing amounts of extracellular Mg (0.5, 0.03, and 0.01 mM MgSO4). In comparison to cells grown in 0.5 mM Mg, cells grown in 0.03 mM Mg exhibited a decrease in TopoII activity, as evidenced by reduced induction of DNA/TopoII cleavable complexes and apoptosis by etoposide and teniposide. Enzyme activity was restored by the readdition of Mg (0.5 and 1.5 mM) in the incubation medium, confirming that this effect was indeed modulated by extracellular Mg. Restriction of Mg to 0.01 mM was associated with a dramatic decrease in TopoII activity resembling that observed in HL-60 cells differentiated by dimethyl sulfoxide treatment. The restriction of Mg, while decreasing enzyme activity, was found to upregulate TopoII protein expression, determined by Western blot analysis. The increase of TopoII protein levels was correlative with the degree of Mg deprivation. Collectively, these results indicate that extracellular levels of Mg may control availability of intracellular Mg, thus affecting the regulation of TopoII activity/expression and downstream processes of cell proliferation and/or differentiation. J. Cell. Biochem. 78:325–333, 2000. © 2000 Wiley-Liss, Inc.

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.

Regulation of topoisomerase II (TOPO II) isozymes  and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO II and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II ( and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II ( + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment.© 1998 by The American Society of Hematology.

Regulation of topoisomerase II (TOPO II) isozymes  and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO II and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II ( and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II ( + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment.© 1998 by The American Society of Hematology.

Tumor cell resistance to inhibitors of topoisomerase II (topo II) is associated frequently with the overexpression of P-glycoprotein (PGP), and strategies to overcome resistance are focused on restoring defects in drug accumulation. Inhibitors of calcium-calmodulin-dependent enzymes sensitize resistant tumor cells to the topo II poison etoposide (VP-16) by enhancing DNA damage and an apoptotic response. In the present study, we have investigated the consequences of buffering intracellular calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra(acetoxy-methyl) ester (BAPTA-AM) on the sensitizing effects of the calmodulin-dependent protein kinase II inhibitor 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-piperazine (KN-62) in etoposide-resistant human leukemia HL-60 (HL-60/ADR0.05) cells. In cells pretreated with 20 microM BAPTA-AM for 2 hr, extracellular ATP failed to trigger intracellular calcium transients, and no effects on the accumulation of VP-16 were apparent. Also, the effect of KN-62 in significantly (P=0.002 to 0.042) enhancing the accumulation of VP-16 in HL-60/ADR0.05 cells was unaffected due to pretreatment with BAPTA-AM. In contrast, pretreatment with BAPTA-AM reduced the DNA damage induced by VP-16, and significantly (P=0.038) reversed the enhancement by KN-62 of VP-16-stabilized topo II-mediated DNA cleavable complex formation. The pretreatment of HL-60/ADR0.05 cells with BAPTA-AM was also associated with the hypophosphorylation of topo IIalpha. Consistent with the ability of BAPTA-AM to circumvent the potentiation by KN-62 of VP-16-induced DNA damage, survival of cells treated with 40 microM VP-16 in the absence of KN-62 and 10 microM VP-16 in the presence of KN-62 was significantly (P=0.026 to 0.031) higher due to BAPTA-AM pretreatment. Results demonstrate that intracellular calcium transients could play a key role in the sensitization of etoposide-resistant tumor cells by inhibitors of calcium-calmodulin-dependent enzymes.

BACKGROUND To test the hypothesis that the cytoprotectant amifostine attenuates the thrombocytopenia produced by carboplatin, the authors performed a randomized trial comparing treatment with carboplatin alone versus the combination of amifostine and carboplatin. METHODS Patients with refractory or carboplatin-sensitive malignancies were randomized to receive either carboplatin, 500 mg/m2 alone or carboplatin, 500 mg/m2 in conjunction with 2 doses of amifostine of 910 mg/m2 each. RESULTS Fifty-five patients with a variety of malignancies were entered on this study. One patient withdrew from each arm prior to the administration of any therapy, leaving 30 evaluable patients treated with carboplatin alone and 23 treated with the combination of amifostine and carboplatin. For 82 cycles of therapy with amifostine plus carboplatin, the median platelet nadir was 127 × 109/L while the median platelet nadir was 88 × 109/L over the 80 courses of therapy with carboplatin alone (P = 0.023). The median platelet nadir after the first cycle of therapy was 144 × 109/L for patients treated with amifostine plus carboplatin and 85 × 109/L for patients treated with carboplatin alone (P = 0.24). The median survival for 9 patients with advanced nonsmall cell lung carcinoma treated with carboplatin alone was 39 weeks whereas the median survival for 12 such patients treated with amifostine plus carboplatin was 52 weeks (P = 0.116). CONCLUSIONS These data support the hypothesis that amifostine attenuates the myelosuppression of carboplatin. Additional studies of amifostine in combination with carboplatin-containing chemotherapy regimens are warranted. Cancer 1997; 80:1134-40. © 1997 American Cancer Society.

Inhibitors of calcium-calmodulin-dependent processes, 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-piperazine KN-62 and trifluoperazine (TFP), at non-cytotoxic concentrations (2 and 5 microM, respectively) enhanced etoposide (VP-16) cytotoxicity in Adriamycin-resistant (HL-60/ADR0.05) cells (3- to > 50-fold). In contrast to TFP, the inhibitor KN-62 was able to reverse resistance in HL-60/ADR0.05 cells at VP-16 concentrations that produced equivalent cytotoxicity in sensitive (HL-60/S) cells. Unlike TFP, the cellular accumulation of VP-16 in the presence of KN-62 was enhanced 1.5- to 2-fold in HL-60/S (MDR1 -ve) and HL-60/ADR0.05 (MDR1 +ve) cells. To achieve equivalent cytotoxicity, levels of VP-16 in the resistant cells were > 4-fold lower in the presence of KN-62 compared with treatment with VP-16 alone. The sensitizing effects of both KN-62 and TFP were due to enhancement (2- to 4-fold) of VP-16-induced topoisomerase II (TOPO II)-mediated DNA cleavable complex formation, and depletion of the 170 kDa (alpha) TOPO II isoform. The DNA damage induced by VP-16 in the presence of KN-62 or TFP resulted in the rapid induction of apoptosis and depletion of cells in "S" phase of the cell cycle. Both 5 microM TFP and 2 microM KN-62 enhanced the phosphorylation of 170 kDa TOPO II 1.6-fold and 1.5-fold, respectively. Results suggest that the inhibitory effect of KN-62 or TFP on calcium-calmodulin-dependent processes may be mechanistically involved in sensitizing resistant cells to VP-16 by enhancing TOPO II-mediated DNA damage.

Tumor cell resistance to doxorubicin (DOX) is usually associated with the overexpression of P-glycoprotein (PGP) in model systems. We have characterized the karyotypic changes in two sublines of HL-60 cells which differ in the induction of differentiation by retinoic acid. The parental sublines, designated HL-60A/S and HL-60Y/S, were selected in increasing concentrations of 0.025-0.1 micrograms/mL DOX. Monosomy 8 in HL-60Y/S was the only karyotypic difference prior to DOX exposure. Both sublines acquired 7q+ markers upon exposure to DOX. In HL-60Y/S, and add(7)(q21) replaced one homologue at 0.025 micrograms/mL DOX, and an add(7)(q32) appeared which replaced the other normal 7 at 0.05 micrograms/mL DOX. The HL-60A/S cells acquired an add(7)(q21) at 0.025 micrograms/mL DOX. The 7q+ abnormalities involved breakpoints in the midregion of 7q. The overexpression of phosphorylated PGP in immunoprecipitates with C-219 antibody was identified in both sublines of DOX-resistant HL-60 cells with 7q+ abnormalities, and this is consistent with the location of mdr-1 sequences to 7q21-21.1. Also, analysis of RNA from parental-sensitive and DOX-resistant sublines by reverse transcriptase-polymerase chain reaction revealed: a) comparable expression of multidrug resistance related protein (MPR) in sensitive and resistant sublines; and b) overexpression of mdr-1 only in the DOX-resistant sublines. Thus, the selection of DOX resistance in two sublines of HL-60 cells which differ in their response to retinoic acid-induced myeloid differentiation is reproducibly associated with overexpression of mdr-1 versus MRP.

Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp). The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells. We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells. Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest. Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues. Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase. These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies. Images Figure 6

In sporadic clear cell renal cell carcinoma (CCRCC), the von Hippel Lindau (VHL) gene is inactivated by mutation or methylation in the majority of primary (P) tumors. Due to differing effects of wild-type (WT) and mutant (MT) VHL gene on downstream signaling pathways regulating angiogenesis, VHL gene status could impact clinical outcome. In CCRCC, comparative genomic hybridization (CGH) analysis studies have reported genetic differences between paired P and metastatic (M) tumors. We thus sequenced the VHL gene in paired tumor specimens from 10 patients to determine a possible clonal relationship between the P tumor and M lesion(s) in patients with CCRCC. Using paraffin embedded specimens, genomic DNA from microdissected samples (>80% tumor) of paired P tumor and M lesions from all 10 patients, as well as in normal tissue from 6 of these cases, was analyzed. The DNA was used for PCR-based amplification of each of the 3 exons of the VHL gene. Sequences derived from amplified samples were compared to the wild-type VHL gene sequence (GeneBank Accession No. AF010238). Methylation status of the VHL gene was determined using VHL methylation-specific PCR primers after DNA bisulfite modification. In 4/10 (40%) patients the VHL gene status differed between the P tumor and the M lesion. As expected, when the VHL gene was mutated in both the P tumor and M lesion, the mutation was identical. Further, while the VHL genotype differed between the primary tumor in different kidneys or multiple metastatic lesions in the same patient, the VHL germline genotype in the normal adjacent tissue was always wild-type irrespective of the VHL gene status in the P tumor. These results demonstrate for the first time that the VHL gene status can be different between paired primary and metastatic tissue in patients with CCRCC.

Topoisomerase II protein is essential for cell proliferation and is known to exist as a phosphoprotein in cells from both lower and higher eukaryotic species. In this paper, we have investigated the phosphorylation of the alpha isozyme of human topoisomerase II. The topoisomerase II alpha protein was phosphorylated predominantly on serine residues in the human tumor cell lines HeLa and NSCLC-3. Two-dimensional tryptic phosphopeptide mapping studies revealed several sites of phosphorylation in vivo, including a major site that was common to topoisomerase II alpha protein from both HeLa and NSCLC-3 cells. To identify sites of phosphorylation, the regulatory C-terminal domain of human topoisomerase II alpha protein was overexpressed in Escherichia coli as a hexahistidine-tagged fusion protein and purified by nickel chelate chromatography. Tryptic phosphopeptide mapping revealed that casein kinase II phosphorylated the C-terminal domain primarily on 2 serine residues in vitro, which were shown to be sites of modification in vivo. Site-directed mutagenesis studies identified these casein kinase II-specific phosphorylation sites as serine 1524 and serine 1376.

Background: Tivantinib, an oral, non-ATP competitive, selective c-MET inhibitor, exhibited antitumor activity in preclinical models. This open-label, phase I, dose-escalation study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of tivantinib in patients with advanced or metastatic solid tumors refractory to standard therapy. Methods: Thirteen dose levels of tivantinib ranging from 10 to 360 mg twice a day were administered to patient cohorts in 21-day cycles (14 days on/7 days off); three active pharmaceutical ingredient forms of tivantinib (amorphous, crystalline A, and crystalline B) were also investigated. Treatment was continued until the occurrence of unacceptable toxicity, tumor progression, patient withdrawal, or death. Results: A total of 79 patients with advanced solid tumors were enrolled. A maximum tolerated dose was not determined. Tivantinib was well tolerated, with mild to moderate toxicities. Two patients discontinued the study drug due to treatment-emergent adverse events. Dose-limiting grade of 3 or more toxicities including leukopenia, neutropenia, thrombocytopenia, vomiting, and dehydration, were observed in 2 patients treated with tivantinib 360 mg twice a day. The rate of absorption of tivantinib peaked approximately 2 to 4 hours after initial dosing, followed by a linear decrease in plasma concentrations. Increases in tivantinib exposure were not dose proportional. There was significant interpatient pharmacokinetic variability; however the clinical safety of tivantinib seemed unaffected. Three patients (3.8%) achieved a partial response and 40 patients (50.6%) maintained stable disease for a median of 19.9 weeks. Conclusions: Tivantinib 360 mg twice a day was well tolerated in patients with refractory advanced solid tumors. The results of this trial warrant further clinical investigation. Clin Cancer Res; 17(24); 7754–64. ©2011 AACR.

Biomarkers that predict response or toxicity to antiangiogenic therapy are sought to favorably inform the risk/benefit ratio. This study evaluated the association of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) genetic polymorphisms with the development of hypertension (HTN) and clinical outcome in metastatic clear cell renal cell carcinoma (MCCRCC) patients treated with sunitinib.Sixty-three MCCRCC patients receiving sunitinib (50 mg 4/2) with available blood pressure (BP) data and germline DNA were retrospectively identified. A panel of candidate VEGF and VEGFR2 single nucleotide polymorphisms (SNPs) were evaluated for associations with the development of hypertension and clinical outcome.VEGF SNP -634 genotype was associated with the prevalence and duration of sunitinib-induced hypertension (as defined by systolic pressure ≥150 mmHg and/or diastolic pressure ≥90 mmHg) in both univariable analysis (P = .03 and .01, respectively) and multivariable analysis, which adjusted for baseline BP and use of antihypertension medication (P = .05 and .02, respectively). Patients with the GG genotype were estimated to have a greater likelihood of being hypertensive during treatment compared with patients with the CC genotype (odds ratio of 13.62, 95% confidence interval [CI] 3.71-50.04). No single VEGF or VEGFR SNPs were found to correlate with clinical outcome. However, the combination of VEGF SNP 936 and VEGFR2 SNP 889 were associated with overall survival after adjustment for prognostic risk group (P = .03).In MCCRCC patients treated with sunitinib, VEGF SNP -634 is associated with hypertension and a combination of VEGF SNP 936 and VEGFR2 SNP 889 genotypes is associated with overall survival.

Trifluoperazine (TFP) is effective in modulating DNA damage/repair in doxorubicin (DOX) treated cells. In the present study we have characterised the resistance phenotype of parental sensitive L1210 mouse leukaemia cells (L1210/S) adapted to grow in the presence of 0.017 microns DOX+5 microM TFP (L1210/DT). Although with prolonged exposure, 0.017 microM DOX alone produced < 35% cell kill in L1210/S cells, similar cytotoxicity was achieved at 0.43 microM DOX in L1210/S cells selected in the presence of 0.017 microM DOX+5 microM TFP. L1210/DT cells were > 30-fold resistant to DOX following a 3 h drug exposure in a soft agar colony assay. In contrast, DOX sensitivity in cells adapted to grow in 5 microM TFP alone was comparable to L1210/S cells. Resistance to other inhibitors of topoisomerase II in L1210/DT cells was > 30-fold to etoposide and > 6-fold to amsacrine. The levels of the 170 kDa and 180 kDa isoforms of topoisomerase II in an immunoblot were comparable between the L1210/S and L1210/DT cells. Cross resistance to vincristine in the L1210/DT cells was accompanied by the overexpression of plasma membrane P-glycoprotein. Although a 1.5-2-fold decrease in accumulation of etoposide and DOX was observed in the L1210/DT cells, drug levels for equivalent DNA damage in the alkaline elution assay were > 5-fold higher in the L1210/DT versus L1210/S cells. No abrogation in the modulating effects of TFP on DOX, VP-16 or amsacrine induced cytotoxicity was apparent in the L1210/DT cells. Results suggest that: (a) TFP in combination with low concentrations DOX can induce the selection of cells with the multidrug resistant phenotype; and (b) characteristics of cells selected for resistance to DOX or DOX plus TFP are comparable. Images Figure 5 Figure 6

CCL2, a chemokine, is expressed in normal human ovarian epithelium but down-regulated in ovarian adenocarcinomas. The association of CCL2 expression with chemotherapy response, invasion and survival outcomes was studied in patients with primary ovarian cancer (OC) and in ovarian cancer cell lines (OCCLs). Tumor specimens (80% tumor) from patients with primary, advanced serous OC obtained at the time of cytoreductive surgery was used to isolate total RNA. The CCL2 gene expression evaluated by RT-PCR was investigated in relation to chemo-response/clinical outcomes in the OC patients and to sensitivity to cisplatin/paclitaxel in the OCCLs. In vitro invasion was measured by matrigel invasion and matrixmetallo-proteinase-9 (MMP-9) zymogram assays. Thirty-seven patients were included. In multivariable analyses that adjusted for the impact of debulking status, the CCL2 mRNA expression was correlated with objective complete response (p = 0.01), chemosensitivity (p = 0.04), and progression-free survival (PFS; p = 0.006). These findings were corroborated in vitro in the OCCLs. The cells expressing higher levels of CCL2 were more sensitive to paclitaxel and cisplatin as compared to those lines expressing lower levels of this chemokine. Up-regulation of CCL2 in the PAT-7 cell line further enhanced the response of these cells to paclitaxel (p = 0.0001) and led to decreased invasion (p = 0.0009). Increased ovarian tumoral expression of CCL2 is associated with improved chemoresponse and survival outcomes, and higher levels of CCL2 in ovarian cancer cell lines are associated with increased chemosensitivity and decreased invasion in vitro.

We recently identified germline methylation of KILLIN, a novel p53-regulated tumor suppressor proximal to PTEN, in >1/3 Cowden or Cowden syndrome-like (CS/CSL) individuals who are PTEN mutation negative. Individuals with germline KILLIN methylation had increased risks of renal cell carcinoma (RCC) over those with PTEN mutations. Therefore, we tested the hypothesis that KILLIN may be a RCC susceptibility gene, silenced by germline methylation. We found germline hypermethylation by combined bisulfite restriction analysis in at least one of the four CpG-rich regions in 23/41 (56%) RCC patients compared to 0/50 controls (P < 0.0001). Of the 23, 11 (48%) demonstrated methylation in the -598 to -890 bp region in respect to the KILLIN transcription start site. Furthermore, 19 of 20 advanced RCC showed somatic hypermethylation upstream of KILLIN, with the majority hypermethylated at more than one CpG island (13/19 vs. 3/23 with germline methylation, P < 0.0001). qRT-PCR revealed that methylation significantly downregulates KILLIN expression (P = 0.05), and demethylation treatment by 5-aza-2'deoxycytidine significantly increased KILLIN expression in all RCC cell lines while only increasing PTEN expression in one line. Furthermore, targeted in vitro methylation revealed a significant decrease in KILLIN promoter activity only. These data reveal differential epigenetic regulation by DNA promoter methylation of this bidirectional promoter. In summary, we have identified KILLIN as a potential novel cancer predisposition gene for nonsyndromic clear-cell RCC, and the epigenetic mechanism of KILLIN inactivation in both the germline and somatic setting suggests the potential for treatment with demethylating agents.

The goal of this study was to evaluate the pharmacokinetics, mass balance, metabolism, routes and extent of elimination, and safety of a single oral dose of (14)C-labeled brivanib alaninate and the safety and tolerability of brivanib after multiple doses in patients with advanced or metastatic solid tumors. This was a two-part, single-center, open-label, single oral-dose (part A) followed by multiple-dose (part B) study in patients with advanced or metastatic solid tumors. In part A, patients received a single dose of [(14)C]brivanib alaninate and in part B patients received 800 mg of nonradiolabeled brivanib alaninate every day. Four patients (two white, two black: two with non-small-cell lung cancer, one with ovarian cancer, and one with renal cell carcinoma) were treated in both parts. The median time to reach the maximal plasma concentration of brivanib was 1 h, geometric mean maximal plasma concentration was 6146 ng/ml, mean terminal half-life was 13.8 h, and geometric mean apparent oral clearance was 14.7 l/h. After a single oral dose of [(14)C]brivanib alaninate, 12.2 and 81.5% of administered radioactivity was recovered in urine and feces, respectively. Brivanib alaninate was completely converted to the active moiety, brivanib, and the predominant route of elimination was fecal. Renal excretion of unchanged brivanib was minimal. Brivanib was well tolerated; fatigue was the most frequent adverse event occurring in all patients and the most frequent treatment-related adverse event in three (75%). The best clinical response in one patient was stable disease; the other three had progressive disease. Brivanib alaninate was rapidly absorbed and extensively metabolized after a single 800-mg oral dose; the majority of drug-related radioactivity was excreted in feces.

Recently several low-grade renal cell tumors, distinct from those recognized by the 2004 World Health Organization classification of renal tumors, have been described. These tumors had similar clinicopathologic features, being low-stage tumors with cystic, tubuloacinar, and/or papillary architecture. The tumor cells were low grade with variable amounts of clear cytoplasm that was positive for cytokeratin 7 (CK7), but negative for CD10. Genetic changes characteristic of clear cell or papillary renal cell carcinoma were not seen in these tumors. We investigated the morphologic, immunohistochemical, and genetic features of 36 additional tumors. Immunohistochemistry was carried out for CK7, carbonic anhydrase 9, α-methylacyl-CoA racemase, CD10, TFE-3, and desmin. Interphase fluorescence in situ hybridization was carried out with centromeric probes for chromosomes 3, 7, 17, and a subtelomeric probe for 3p25. Sequencing of von Hippel-Lindau gene and analysis of the methylation status of the promoter region was also carried out in 2 tumors. Thirty-six tumors from 33 patients (mean age: 60.4 , range: 26 to 88; 17 men and 16 women) were studied. Three patients had bilateral tumors and 1 patient had von Hippel-Lindau disease. Follow-up was available in 60% (20/33) of the patients for a mean of 27.4 (range 1 to 85) months. No patient had evidence of the disease after surgery except for the patient with von Hippel-Lindau disease, who was alive with stable disease in the contralateral kidney. All 36 tumors were small (mean size 2.4 cm; range 0.9 to 4.5 cm) and low stage (pT1). The majority was cystic and had prominent fibrous capsule and stroma. The tumors were composed of variable amount of cysts, papillae, tubules, acini, and solid nests. The most characteristic histologic features were branching tubules and acini and anastomosing clear cell ribbons with low-grade nuclei. All tumors were strongly positive for CK7 and variably positive for CA9, but largely negative for CD10, and negative for α-methylacyl-CoA racemase and TFE-3. All but 1 tumor had no gains of chromosomes 7 and 17 and deletion of 3p. Only 1 tumor had low copy number gains of chromosomes 7 and 17. VHL gene mutation and promoter methylation were negative in 2 tumors analyzed. We show that these tumors, which we term as "clear cell tubulopapillary renal cell carcinoma," constitute a unique subtype in the spectrum of renal epithelial neoplasia based on their characteristic morphologic and immunohistochemical features.

Angiogenesis is central to the growth of normal tissues and tumors. Inhibiting this pathway has been a strategy for drug development for tumors not responsive to most agents used in chemotherapy. Notably, signaling mediated by vascular endothelial growth factor (VEGF) is a key target because aberrant signaling via this pathway is frequently associated with neoangiogenesis in tumors. The drug-discovery effort to blunt VEGF signaling has led to the approval of bevacizumab and several receptor tyrosine kinase inhibitors (TKIs) that have shown efficacy in the clinical management of breast, colorectal, lung, and kidney cancer. Understanding the genetic variability in VEGF and VEGF receptor has led to identifying genotypic variations (single nucleotide polymorphisms [SNPs]) associated with treatment outcome and toxicity. Notably, identification of SNPs in VEGF associated with angiogenesis inhibitor treatment-induced hypertension and outcome provides exciting opportunities for personalized medicine to improve outcome and reduced toxicity with these novel TKIs.

The overexpression of P-glycoprotein (PGP) and alterations in DNA topoisomerase II (TOPO II) were evaluated in mouse leukemia P388 cells selected in vivo for mitoxantrone (MTT) resistance (P388/MTT) and compared to doxorubicin (DOX) resistant (P388/DOX) or vincristine (VCR) resistant (P388/VCR) models. Among a panel of TOPO II inhibitors which included etoposide (VP-16), DOX, MTT and 4'-[(9-acridinyl)-amino]methanesulfon-m-anisidide (m-AMSA), the relative resistance compared to parental sensitive P388/S cells was: P388/DOX greater than P388/MTT greater than P388/VCR. All the resistant sublines exhibited minimal cell kill (less than 20%) at vincristine concentrations greater than 100-fold the IC50 for P388/S cells. In a soft-agar colony-forming assay, the modulation of cytotoxicity in P388/MTT cells by the calmodulin inhibitor trifluoperazine following a 3-hr drug treatment demonstrated a marked potentiation in cell kill with MTT, VP-16, DOX and m-AMSA but not VCR. Immunoblotting data revealed that while PGP was not detectable in P388/S cells, the overexpression of PGP was apparent in P388/MTT cells and the relative expression between the resistant sublines was: P388/DOX greater than P388/MTT greater than P388/VCR. Although the amount and DNA cleavage activity of TOPO II in nuclear extracts from P388/VCR cells were comparable to those in P388/S cells, they were markedly lower in both P388/DOX and P388/MTT cells. However, decatenation activity of TOPO II in nuclear extracts was comparable between the sensitive (P388/S) and resistant sublines (P388/MTT, P388/DOX, and P388/VCR). Results from the present study demonstrated that P388 cells selected for resistance to mitoxantrone exhibit changes in TOPO II and overexpression of PGP similar to P388/DOX cells, while vincristine resistant cells only overexpress PGP. Since therapeutic strategies are primarily designed to interfere with PGP-mediated drug efflux, the choice of agents for modulating resistance in tumors which overexpress PGP versus tumors which overexpress PGP with altered TOPO II could be different.

Antiproliferative effects of proteasome inhibitors are suggested to be primarily due to effects on nuclear factor-kappaB (NF-kappaB)-dependent pathways and the induction of apoptosis. The objective of this study was to elucidate the mechanistic basis for the antiproliferative effects of the proteasome inhibitor, bortezomib, in human clear cell renal cell cancer cells (CCRCC).von Hippel Lindau (VHL) mutation/methylation status and cytotoxic response to bortezomib was determined in a panel of CCRCC cell lines. Effects on target protein/gene expression and the role of p53 in bortezomib-mediated cytotoxicity, inhibition of proteasome activity, survivin transcript and protein expression as well as induction of p21 expression was determined in CCRCC that differed in their intrinsic sensitivity to bortezomib.VHL status was not associated with cytotoxic response to bortezomib treatment. Cytotoxicity in cell lines that differed in intrinsic sensitivity to bortezomib correlated with sustained inhibition of proteasome activity, survivin expression and induction of p21 expression. Stable down-regulation of p53 expression by siRNA led to attenuation of bortezomib effects, survivin down-regulation and p21 induction, suggesting that cellular effects are p53-dependent.These results demonstrate that the antiproliferative effects of bortezomib in CCRCC cells are VHL independent and dependent on pathways regulated by p53.

In kidney cancer, the von Hippel–Lindau protein (pVHL) is an integral part of an E3 ubiqutin ligase complex that targets the degradation of hypoxia inducible factor-1α by the 26 S proteasome under normal oxygen levels. Further, the 26 S proteasome has been shown to play a major physiological role in apoptosis, primarily by regulating the cellular level of p53 and NFκB. Our studies in a panel of wild-type or mutant VHL clear cell renal cell carcinoma (RCC) cell lines demonstrated that VHL status was not associated with cytotoxic response to bortezomib (PS-341) treatment. Cytotoxicity was correlated with downregulation of proteasome activity, survivin expression and induction of p21 expression. Downregulation of p53 expression by siRNA led to attenuation of PS-341 effects, survivin downregulation, and p21 induction, suggesting that cellular effects are p53-dependent. These results suggest that in vitro, the antiproliferative effects of PS-341 in RCC cells are p53-dependent. Despite these data, two multi-institutional phase II studies evaluating the clinical activity and safety of the proteosome inhibitor bortezomib (PS-341) in RCC have failed to demonstrate the clinical utility of this agent in RCC. Since the proteasome pathway remains of importance in RCC biology, future studies understanding the relationship between pVHL, the proteasome complex, and NFκB could be helpful in designing clinical trials pursuing dual inhibition of vascular endothelial growth factor and proteasome signaling pathways.

The von Hippel-Lindau (VHL) gene is often inactivated (by mutation or promoter hypermethylation) in renal cell carcinoma but the relation to therapeutic outcome is unclear.Patients with metastatic clear cell renal cell carcinoma with available baseline tumor samples who received vascular endothelial growth factor targeted therapy were included in analysis. Patient characteristics, VHL gene status and clinical outcome were documented. Our primary end point was to test for response rate in relation to VHL inactivation. Progression-free survival and overall survival in relation to VHL status were investigated as secondary end points.A total of 123 patients were evaluable. Response rate, median progression-free survival and median overall survival were 37% (95% CI 28-46), 10.8 (95% CI 7.7-14.8) and 29.8 (CI not estimable) months, respectively. Patients with VHL inactivation had a response rate of 41% vs 31% for those with wild-type VHL (p = 0.34). Patients with loss of function mutations (frameshift, nonsense, splice and in-frame deletions/insertions) had a 52% response rate vs 31% with wild-type VHL (p = 0.04). On multivariate analysis the presence of a loss of function mutation remained an independent prognostic factor associated with improved response. Progression-free survival and overall survival were not significantly different based on VHL status.To our knowledge this is the largest analysis investigating the impact of VHL inactivation on the outcome of vascular endothelial growth factor targeted agents in metastatic renal cell carcinoma. We did not find a statistically significant increase in response to vascular endothelial growth factor targeted agents in patients with VHL inactivation. Loss of function mutations identified a population of patients with a greater response. Investigation of downstream markers is under way.

The potentiation of topoisomerase (topo)-I-induced apoptosis by proteasome inhibitors is dependent on the treatment sequence, but not on NF-kappaB. In this study, alternate mechanisms modulating apoptosis induced with the topo I-targeting drug, SN-38, when followed by the proteasome inhibitor bortezomib (PS-341) were investigated.Human non-small cell lung carcinoma (NSCLC-3) cells transfected with a control vector (NSCLC-3/neo) or a vector containing dominant negative IkappaBalpha (NSCLC-3/mIkappaBalpha) were treated with SN-38 for 1 h followed by PS-341 for 4 h (SN-38 --PS-341), or with either drug alone. The functional role of the anti-apoptotic protein survivin was tested using NSCLC-3 transfected with myc-tagged wild-type (NSCLC-3/myc-survivin), or dominant negative mutant T34A survivin (NSCLC-3/myc-T34A).In NSCLC-3/neo or NSCLC-3/mIkappaBalpha cells, treatment with SN-38 --PS-341 led to down-regulation of the survivin transcript and protein, enhanced apoptosis and reduced (3-fold) survival compared to SN-38 or PS-341 alone. In contrast to the cells transfected with wild-type survivin, or the control NSCLC-3/neo, those cells transfected with mutant survivin and treated with SN-38 --PS-341 exhibited enhanced caspase 9 activity (2-fold), caspase 3 (2- to 3-fold) activity and cytotoxicity compared to the NSCLC-3/neo cells.In contrast to inhibition of NF-kappaB activity, down-regulation of the anti-apoptotic survivin was correlated with modulation of the sequence-dependent synergistic effects of PS-341 in SN-38-induced apoptosis.

Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-κB–independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I–targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38→PS-341). The p53-dependent sensitization of DNA damage–induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341→SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341→SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38→PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 + M phase of the cell cycle in SN-38→PS-341–treated cells compared with PS-341→SN-38–treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3σ and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3σ or survivin in regulating the divergent function of p53 in response to SN-38→PS-341 and PS-341→SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms by which inhibition of proteasome activity modulates DNA damage–induced apoptosis via a p53-dependent pathway. [Mol Cancer Ther 2005;4(12):1880–90]

Renal cell carcinomas (RCC) commonly retain wild-type but functionally inactive p53, which is repressed by an unknown dominant mechanism. To help reveal this mechanism, we screened a diverse chemical library for small molecules capable of restoring p53-dependent transactivation in RCC cells carrying a p53-responsive reporter. Among the compounds isolated were derivatives of 9-aminoacridine (9AA), including the antimalaria drug quinacrine, which strongly induced p53 function in RCC and other types of cancer cells. Induction of p53 by these compounds does not involve genotoxic stress and is mediated by suppression of NF-kappaB activity. In contrast to agents that target IkappaB kinase 2, 9AA and quinacrine can effectively suppress both basal and inducible activities of NF-kappaB, representing inhibitors of a previously undescribed type that convert NF-kappaB from a transactivator into a transrepressor, leading to accumulation of inactive nuclear complexes with unphosphorylated Ser-536 in the p65/RelA subunit. p53 function in RCC can be restored by ectopic expression of a superrepressor of IkappaB as effectively as by 9AA-derived compounds. These findings suggest that the complete or partial repression of p53 observed in many tumors can be the result of constitutive activation of NF-kappaB. The results demonstrate, in principle, the possibility to kill cancer cells selectively through simultaneous inhibition of NF-kappaB and activation of p53 by a single small molecule and suggest anticancer applications for the well known antimalaria drug quinacrine.

Purpose: This study intended to determine the maximum tolerated dose, safety, pharmacokinetic variables, clinical response, and pharmacodynamic markers of daily s.c. administration of Angiozyme. Patients and Methods: Patients with refractory solid tumors were enrolled in a dose escalation and expanded cohort design. Dose escalation involved cohorts of patients at doses of 10, 30, 100, or 300 mg/m2/d for 29 days. A second component enrolled 15 additional patients at a daily dose of 100 mg/m2. Patients were eligible to continue on therapy until disease progression. Results: Thirty-one patients were enrolled and 28 were evaluable (range, 29–505 days; median, 89.5 days). A maximum tolerated dose was not defined by toxicity but rather by the maximal deliverable dose of 300 mg/m2/d. Grade 1 to 2 injection site reactions were the most common toxicities. One patient in the 300 mg/m2 group experienced a reversible grade 3 injection site reaction. Angiozyme showed dose-dependent plasma concentrations with good bioavailability. Surrogate markers showed Angiozyme localization in tumor biopsies and a significant increase in serum von Willebrand factor antigen, a marker for endothelial cell dysfunction. Although Angiozyme-reactive antibody production was noted for some patients, no antibody-related adverse events were noted. Seven of 28 (25%) evaluable patients had stable disease for ≥6 months, with the longest treatment duration of ≥16 months. Two patients (nasopharyngeal carcinoma and melanoma) showed minor responses. Conclusion: Angiozyme was well tolerated with satisfactory pharmacokinetic variables for daily s.c. dosing. Results have provided the basis for subsequent clinical trials of this first-of-class biologically targeted therapeutic.

The hereditary prostate cancer 1 (HPC1) allele maps to the RNASEL gene encoding a protein (RNase L) implicated in the antiviral activity of interferons. To investigate the possible role of RNase L in apoptosis of prostate cancer cells, we decreased levels of RNase L by severalfold in the DU145 human prostate cancer cell line through the stable expression of a small interfering RNA (siRNA). Control cells expressed siRNA with three mismatched nucleotides to the RNase L sequence. Cells deficient in RNase L, but not the control cells, were highly resistant to apoptosis by the RNase L activator, 2′,5′-oligoadenylate (2-5A). Surprisingly, the RNase L-deficient cells were also highly resistant to apoptosis by combination treatments with a topoisomerase (Topo) I inhibitor (camptothecin, topotecan, or SN-38) and tumor necrosis factor-related apoptosis-inducing ligand [TRAIL (Apo2L)]. In contrast, cells expressing siRNA to the RNase L inhibitor RLI (HP68) showed enhanced apoptosis in response to Topo I inhibitor alone or in combination with TRAIL. An inhibitor of c-Jun NH2-terminal kinases reduced apoptosis induced by treatment with either 2-5A or the combination of camptothecin and TRAIL, thus implicating c-Jun NH2-terminal kinase in the apoptotic signaling pathway. Furthermore, prostate cancer cells were sensitive to apoptosis from the combination of 2-5A with either TRAIL or Topo I inhibitor, whereas normal prostate epithelial cells were partially resistant to apoptosis. These findings indicate that RNase L integrates and amplifies apoptotic signals generated during treatment of prostate cancer cells with 2-5A, Topo I inhibitors, and TRAIL.

The anti-epileptic diphenylhydantoin (DPH; Dilantin®) selectively enhances the in vitro cytotoxicity of vinca microtubule poisons in both parent sensitive and multi-drug resistant (MDR) human tumor cells. The in vivo clinical activity of this combination has not been fully evaluated. Purpose: To determine the maximum tolerated dose (MTD), dose-limiting toxicities, and preliminary antitumor activity of the combination of intravenous (IV) bolus vinorelbine (VRL) and oral diphenylhydantoin (DPH) in patients (pts) with refractory solid tumors. Methods: Cohorts of 3–6 pts with refractory cancer were treated with escalating doses of weekly IV bolus VRL (I –20.0 mg/m2; II –22.5 mg/m2; III –25.0 mg/m2; IV –27.5 mg/m2; V –30.0 mg/m2; VI –32.5 mg/m2) combined with a fixed oral dose of DPH (400 mg/day) until MTD or progression. During each 35 day cycle, pts received DPH 400 mg/day administered orally on Days −6 to Day +22 and weekly IV bolus infusion of VRL on Days +1, +8, +15, and +22. The cohort treated at the MTD was expanded to further define toxicity. Results: A total of 25 evaluable pts. (9 men; 16 women) were treated with VRL and DPH at dose levels I (n = 5), II (n = 3), III (n = 2), IV (n = 3), V (n = 7) and VI (n = 5) in 5 week cycles over a 16 month period. Dose limiting toxicity occurred at dose level VI (VRL 32.5 mg/m2) and included grade 3 leukopenia (n = 2), grade 3 neutropenia (n = 1) and grade 4 neutropenia (n = 1) occurring within the first cycle of treatment. There were no responses, however 9 pts had stable disease of variable duration (8–56 weeks) and received a median of 2 cycles of treatment (range 2–14). Conclusion: Intravenous bolus administration of VRL and oral administration of a fixed dose of DPH was well tolerated according to the schedule reported here. Although there were no responses, several patients had prolonged disease stabilization. The recommended phase II dose of VRL when used in this combination is 30 mg/m2.

Limited information is available on the correlation of telomerase activity and the clinical and pathological characteristics, in patients with renal cell carcinoma (RCC). Telomerase repeat amplification protocol (TRAP) was used to measure telomerase activity in frozen RCC specimens from partial/radical nephrectomies performed between 1987 and 1991. Presence of tumor tissue was verified by a pathologist using hematoxylin and eosin stained sections. RNA was measured to ensure the presence of intact protein necessary for telomerase expression. Data on demographics, tumor type, and stage at presentation, local recurrence, distant metastasis, disease-free survival (DFS), and overall survival (OS) was collected, and telomerase activity was correlated with each of these variables. Forty-nine of 67 patients (73%) were telomerase positive (+ve). Gender and stage were the only variables that appeared to be associated with telomerase positivity. Tumors were telomerase +ve in 12/21 females (57 %) vs. 37/46 males (80%) (P = 0.07). Tumors were telomerase +ve in 85% of Stage IV, 76% of Stage III, and 70% of Stage I/II patients (P = 0.12). Five-year survival was 0% for Stage IV, 57% for Stage III, and 77% for Stage I/II patients (P < 0.001), DFS 54% for stage III and 84% for Stage I/II patients (P = 0.05). Telomerase activity, however, was not related to survival in either univariate or multivariate analysis. In patients with telomerase +ve tumors 5-year survival was 55%, and with telomerase -ve tumors 58% (P = 0.56). Stage was the only variable associated with OS or DFS in clear cell RCC patients. In patients with advanced disease, there is a high incidence of telomerase positivity was found, within this limited sample, however, no correlation with survival was found.

We previously demonstrated that KN-62, an inhibitor of calcium calmodulin-dependent enzymes, sensitizes human leukemia HL-60 cells resistant to topoisomerase II-targeting drugs. The objective of this study was to determine pathways of apoptosis downstream of DNA damage induced by KN-62 co-treatment with VP-16.HL-60/Y/DOX0.05 cells were treated with VP-16, KN-62, or VP-16 + KN-62. Following treatment, cells were assayed for c-IAP1, c-IAP2 and XIAP protein expression, as well as caspase activation, cytochrome c release and PARP cleavage.Baseline c-IAP1 protein levels were 2-fold higher in HL60 cells selected for resistance to doxorubicin compared to the parent sensitive line. VP-16 and KN-62 co-treatment was associated with caspase activation via the mitochondrial pathway and significant reductions (p = 0.002) in c-IAP1 protein expression but not with c-IAP2 or XIAP.These data suggest that KN-62 co-treatment sensitizes doxorubicin-resistant cells to VP-16-induced apoptosis by enhancing caspase activity and reducing c-IAP1 expression.

Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.

Telomerase is a ribonucleoprotein polymerase that adds telomeric repeats to chromosome ends. This enzyme is deficient in the majority of normal somatic cells, but often is reactivated during tumorigenesis. In the current study, the authors examined telomerase activity in human American Joint Committee on Cancer Stage II colorectal carcinomas and correlated it with traditional prognostic indicators and disease outcome.The telomerase repeat amplification protocol (TRAP) was employed to determine telomerase activity in 122 surgical specimens (from 77 male and 45 female patients) of human Stage II colorectal carcinoma. The primary site of the tumor was the colon in 52 cases and the rectum in 70 cases. Telomerase activity was correlated with traditional prognostic indicators such as gender, age, T classification, tumor size, tumor grade, and disease outcome (overall survival and disease-free survival). The Median follow-up for patients who still were alive was 5.8 years.Telomerase activity was detected in 80% of the tumors (98 of 122 tumors). Telomerase-positive patients differed from telomerase-negative patients in that they tended to be female (41% vs. 21%; P = 0.1), presented with primary tumors of the colon more frequently (49% vs. 17%; P = 0.01), and had a higher T classification (T(4)) (62% vs. 38%; P = 0.04). Univariate and multivariate analyses demonstrated a correlation between telomerase activity and disease-free survival (P = 0.05).Although a large percentage of Stage II colorectal carcinoma samples were positive for telomerase activity, the prognosis for patients with telomerase-negative tumors was found to be worse than that for patients with telomerase-positive tumors.

Activation of signaling pathways following DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. NF-kappaB, a major regulator of the stress response and a negative regulator of apoptosis is often activated following treatment with topoisomerase poisons. Since activation of NF-kappaB is generally considered to relay an anti-apoptotic signal, inactivation of this signaling molecule is considered to represent an important strategy to improve therapeutic efficacy. Although this strategy seems to be effective in some model systems, our results in human non-small cell lung cancers differed. In this review we will discuss the role of NF-kappaB in mediating topoisomerase poison-induced DNA damage and apoptosis and the consequence of inhibiting its activity. Newer insights about the importance of proteasome inhibitors and anti-apoptotic genes in topoisomerase poison-induced signaling mechanisms leading to apoptosis will also be reviewed. The knowledge obtained from these studies may be useful for translation to a clinical setting for development of more effective therapeutic strategies.