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Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2(-/-) cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia-telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2(-/-) cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia-deficient tumors by their sensitization to DNA cross-linking agents is discussed., (©2013 AACR.)

The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 μmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy., (©2011 AACR.)

The recent approval of bevacizumab (Avastin), a humanized anti-vascular endothelial growth factor (VEGF) monoclonal antibody, in combination with chemotherapy for the treatment of patients with metastatic colorectal cancer, has provided proof of principle of the efficacy of antiangiogenic strategies for cancer therapy. The activity of bevacizumab is primarily attributed to its ability to inhibit endothelial cell survival. Whether anti-VEGF strategies may also have a direct effect on cancer cell survival is poorly understood. We show that serum-starved colon cancer cells differentially respond to autocrine production of VEGF with the induction of hypoxia inducible factor-1 alpha (HIF-1 alpha) and survival under hypoxic conditions. Inhibition of VEGF or VEGF receptor 2 (VEGFR2)/KDR, but not VEGFR1/Flt-1, was sufficient to abrogate VEGF-mediated induction of HIF-1 alpha and survival in sensitive HCT116, but not in resistant HT29, colon cancer cells. These results provide evidence that a VEGF/KDR/HIF-1 alpha autocrine loop differentially mediates survival of hypoxic colon cancer cells, and they suggest that colon cancer cells may be intrinsically sensitive or resistant to anti-VEGF strategies, which may determine the therapeutic efficacy of bevacizumab.

The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1alpha and HIF-1beta proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1alpha and HIF-1beta proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1alpha and HIF-1beta proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-kappaB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.

By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIalpha, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S6-substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIalpha, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O6-substituted guanine analogues, O6-benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells.

We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

We have shown previously that the camptothecin analogue topotecan (TPT), a topoisomerase I (Top 1) poison, inhibits hypoxia-inducible factor 1 (HIF-1) transcriptional activity and HIF-1alpha protein accumulation in hypoxia-treated U251 human glioma cells. In this article, we demonstrate that TPT does not affect HIF-1alpha protein accumulation but inhibits its translation. In addition, we demonstrate that Top 1 is required for the inhibition of HIF-1alpha protein accumulation by TPT as shown by experiments performed using camptothecin-resistant cell lines with known Top 1 alterations. Experiments performed with aphidicolin indicated that TPT inhibited HIF-1alpha protein accumulation in the absence of DNA replication. DNA-damaging agents, such as ionizing radiation and doxorubicin, did not affect HIF-1alpha protein accumulation. Ongoing transcription was essential for the inhibition of HIF-1alpha protein accumulation by TPT. Our results demonstrate the existence of a novel pathway connecting Top 1-dependent signaling events and the regulation of HIF-1alpha protein expression and function. In addition, our findings dissociate the cytotoxic activity of TPT from the inhibition of the HIF-1 pathway and raise the possibility of novel clinical applications of TPT aimed at targeting HIF-1-dependent responses.

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation. HIF-1 has been implicated in the regulation of genes involved in angiogenesis [e.g., vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase] and anaerobic metabolism (e.g., glycolytic enzymes). HIF-1 is essential for angiogenesis and is associated with tumor progression. In addition, overexpression of HIF-1 alpha has been demonstrated in many common human cancers. Therefore, HIF-1 is an attractive molecular target for development of novel cancer therapeutics. We have developed a cell-based high-throughput screen for the identification of small molecule inhibitors of the HIF-1 pathway. We have genetically engineered U251 human glioma cells to stably express a recombinant vector in which the luciferase reporter gene is under control of three copies of a canonical hypoxia-responsive element (U251-HRE). U251-HRE cells consistently expressed luciferase in a hypoxia- and HIF-1-dependent fashion. We now report the results of a pilot screen of the National Cancer Institute "Diversity Set," a collection of approximately 2000 compounds selected to represent the greater chemical diversity of the National Cancer Institute chemical repository. We found four compounds that specifically inhibited HIF-1-dependent induction of luciferase but not luciferase expression driven by a constitutive promoter. In addition, these compounds inhibited hypoxic induction of VEGF mRNA and protein expression in U251 cells. Interestingly, three compounds are closely related camptothecin analogues and topoisomerase (Topo)-I inhibitors. We show that concomitant with HIF-1 and VEGF inhibition, the activity of the Topo-I inhibitors tested is associated with induction of cyclooxygenase 2 mRNA expression. The luciferase-based high-throughput screen is a feasible tool for the identification of small molecule inhibitors of HIF-1 transcriptional activation. In addition, our results suggest that altered Topo-I function may be associated with repression of HIF-1-dependent induction of gene expression.

A sensitive DNA dot-blot assay for repetitive human DNA sequences was developed and applied to the quantitative determination of spontaneous metastases of a human melanoma in various tissues of nude mice. The assay was useful for defining the time course and pattern of tissue distribution of metastatic cells as well as for assessing response to therapy. The methodology is relatively simple, can be performed using nonradioactive DNA probes, and should be broadly applicable to studies of metastasis of human tumors in nude mice.

In vivo studies aimed at therapy of spontaneous human tumor metastases have been hampered by the lack of practical experimental models. The LOX amelanotic melanoma model described here represents a transplantation model which rapidly and reproducibly results in spontaneous pulmonary metastasis following s.c. inoculation into athymic mice. Pulmonary lesions can be detected using a simple bioassay procedure which is useful for estimation of metastatic cell killing. Using this model we demonstrate that systemic therapy with cyclophosphamide or dacarbazine can produce metastatic cell killing consistent with complete eradication of established pulmonary metastases. This model may also prove useful for future experimental therapeutic studies aimed at prevention of metastases by manipulating tumor staging interval and treatment schedule.

The applicability of a human tumor colony-forming assay to drug screening was investigated in terms of feasibility, validity, and potential for discovering new antitumor drugs. Feasibility was addressed in a pilot study during which basic methods, appropriate assay quality controls, and a standardized protocol for screening were developed. Considerable variability was noted in the availability and colony growth of different tumor types. The majority of the evaluable experiments utilized breast, colorectal, kidney, lung, melanoma, or ovarian tumors. For many tumor types, little evidence of growth was observed, or only rare specimens formed colonies. Colony-forming efficiencies ranged from 0.05 to 0.11% for the six most useful tumors listed above. A set of quality control measures was developed to address technical problems inherent in the assay. Testing of standard agents in the pilot study established that most of these agents could be detected as active. However, it also identified three assay limitations: compounds requiring systemic metabolic activation are inactive; medium constituents may block the activity of certain antimetabolites; and compounds without therapeutic efficacy may be positive in the assay. The assay categorized nontoxic clinically ineffective agents as true negatives with 97% accuracy. Of 79 compounds which were negative in the current National Cancer Institute prescreen (leukemia P388), 14 were active in the assay. Several demonstrated outstanding in vitro activity and are structurally unrelated to compounds already in development or in clinical trials. A subset of these active compounds were found to lack activity in a P388 in vitro colony-forming assay. This indication of differential cytotoxicity to human tumor cells makes this subset of compounds particularly interesting as antitumor drug leads. The demonstrated sensitivity to most standard agents, discrimination of nontoxic compounds, reproducibility of survival values within assays and between laboratories, and evidence of ability to identify active compounds which were negative in the in vivo prescreen suggest that the human tumor colony-forming assay may be a valuable tool for antitumor drug screening. However, because of technical limitations inherent in the current assay methodology, this must be confined to selected tumor types and limited to screening on a moderate scale.

The propagation efficiencies, growth patterns, histological appearances, and roentgenographic demonstration of tumors derived from six continuous human pulmonary tumor cell lines implanted intrathoracically (i.t.) and intrabronchially (i.b.) were compared with the conventional s.c. implantation method at three different tumor cell inocula (N = 184, i.b.; N = 185, i.t.; N = 180, s.c.). A tumor-related mortality of 100% was noted when the six different human lung tumor cell lines, including A549 adenocarcinoma, NCI-H125 adenosquamous carcinoma, NCI-H460 large cell undifferentiated carcinoma, NCI-H69 small cell carcinoma, and NCI-H358 and NCI-H322 bronchioloalveolar cell carcinomas, were implanted i.b. at a 1.0 x 10(6) tumor cell inoculum. A similar (92%) tumor-related mortality was observed when these same lung tumor cell lines were implanted i.t. at a 1.0 x 10(6) tumor cell inoculum (P greater than 0.10), whereas minimal (5%) tumor-related mortality was noted when cells from the six different cell lines were implanted s.c. (P less than 0.001). In addition, a dose-dependent, tumor-related mortality was noted for either i.t. or i.b. implantation when lower (1.0 x 10(5) or 1.0 x 10(4] tumor cell inocula were employed. Histological characteristics and growth patterns of tumors propagated employing the three implantation techniques were closely comparable for all three propagation methods and, in all instances, histological appearances of the tumors were representative of the current tumor cell lines from which they were derived. Approximately 30% of the lung tumors propagated i.t. grew in the chest wall and/or in the lung parenchyma as well as in the pleural space. In contrast, tumors propagated i.b. grew predominantly in the lung parenchyma. When five nonpulmonary human tumor cell lines (including U251 glioblastoma, LOX amelamontic melanoma, HT-29 colon adenocarcinoma, OVCAR 3 ovarian adenocarcinoma, and adriamycin-resistant MCF-7 breast adenocarcinoma) were propagated i.b. or i.t., there was considerable site-specific variability in tumor-related mortality depending on the tumor type. These data demonstrate that both the i.b. and i.t. models should be useful for the in vivo propagation and study of certain human pulmonary and nonpulmonary carcinomas as well as being advantageous for future studies of cancer biology and developmental therapeutics.

The cytotoxic activities of three new synthetic catechol analogues, beta-[(p-hydroxyphenyl)amino]alanine (Compound 1), N delta-(p-hydroxyphenyl)ornithine (Compound 2), and N delta-(m-hydroxyphenyl)ornithine (Compound 3), were determined against 10 human melanoma and 5 nonmelanoma cell lines. Activities of L-DOPA and 3,4-dihydroxybenzylamine were also measured. Dose-response curves were obtained and concentrations in micrograms/ml required to give 90% inhibition of colony formation (IC90) were calculated. Using a cutoff IC90 of less than 2.5 as a definition of activity. Compound 2 was active in 6 of 10 melanoma and 0 of 5 nonmelanoma cell lines while both Compound 1 and L-DOPA methyl ester were active in 1 of 10 melanomas and 0 of 5 nonmelanomas. Compound 3 was inactive in all cell lines and all IC90 values exceeded 100. 3,4-Dihydroxybenzylamine was active in 3 of 8 melanomas and 1 of 5 nonmelanomas. Regression analysis of IC90 values for Compound 2 and tyrosinase levels in the 15 cell lines yielded a correlation coefficient of 0.93 (P less than 0.001). By contrast, a similar analysis for 3,4-dihydroxybenzylamine gave a correlation coefficient of 0.17 (P greater than 0.05). Spectrophotometric data indicated that Compounds 1 and 2 were oxidized by tyrosinase to quinones. Cytotoxic activity was blocked by the tyrosinase inhibitor phenylthiocarbamide. Since the rates of activation of Compounds 1 and 2 were identical, the higher activity of Compound 2 was probably due to its higher lipophilicity and greater intracellular accumulation. Compounds 1 and 2 exhibited greater potency and selectivity against malignant melanoma than did the natural product L-DOPA methyl ester.

A major impediment to the study of human lung cancer pathophysiology, as well as to the discovery and development of new specific antitumor agents for the treatment of lung cancer, has been the lack of appropriate experimental animal models. This paper describes a new model for the propagation of human lung tumor cells in the bronchioalveolar regions of the right lungs of athymic NCr-nu/nu mice via an intrabronchial (i.b.) implantation procedure. Over 1000 i.b. implantations have been performed to date, each requiring 3 to 5 min for completion and having a surgery-related mortality of approximately 5%. The model was used successfully for the orthotopic propagation of four established human lung cancer cell lines including: an adenosquamous cell carcinoma (NCI-H125); an adenocarcinoma (A549); a large cell undifferentiated carcinoma (NCI-H460), and a bronchioloalveolar cell carcinoma (NCI-H358). When each of the four cell lines was implanted i.b. using a 1.0 X 10(6) tumor cell inoculum, 100 +/- 0% (SD) tumor-related mortality was observed within 9 to 61 days. In contrast, when the conventional s.c. method for implantation was used at the same tumor cell inoculum, only minimal (2.5 +/- 5%) tumor-related mortality was observed within 140 days (P less than 0.001). Similarly, when a 1.0 X 10(5) or 1.0 X 10(4) cell inoculum was used, a dose-dependent, tumor-related mortality was observed when cells were implanted i.b. (56 +/- 24% or 25 +/- 17%) as compared with the s.c. method (5 +/- 5.7% or 0.0 +/- 0%) (P less than 0.02 and P less than 0.05, respectively). Most (greater than 90%) of the lung tumors propagated by i.b. implantation were localized to the right lung fields as documented by necropsy and/or high-resolution chest roentgenography techniques which were developed for these studies. The intrapulmonary model was also used for establishment and propagation of xenografts derived directly from enzymatically digested, fresh human lung tumor specimens obtained at the time of diagnostic thoracotomy and representing all four major lung cancer cell types as well as a bronchioloalveolar cell carcinoma. Approximately 35% (10 of 29) of the fresh primary human lung tumor specimens and 66% (2 of 3) of tumors metastatic to the lung were successfully propagated i.b. at a 1.0 X 10(6) tumor cell inoculum, whereas only 20% (1 of 5) of the specimens were successfully grown in vivo via the s.c. route from a 1.0 X 10(7) tumor cell inoculum.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously described the application of an automated microculture tetrazolium assay (MTA) involving dimethyl sulfoxide solubilization of cellular-generated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-formazan to the in vitro assessment of drug effects on cell growth (M.C. Alley et al., Proc. Am. Assoc. Cancer Res., 27:389, 1986; M.C. Alley et al., Cancer Res. 48:589-601, 1988). There are several inherent disadvantages of this assay, including the safety hazard of personnel exposure to large quantities of dimethyl sulfoxide, the deleterious effects of this solvent on laboratory equipment, and the inefficient metabolism of MTT by some human cell lines. Recognition of these limitations prompted development of possible alternative MTAs utilizing a different tetrazolium reagent, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H- tetrazolium hydroxide (XTT), which is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings, therefore eliminating a solubilization step and shortening the microculture growth assay procedure. Most human tumor cell lines examined metabolized XTT less efficiently than MTT; however, the addition of phenazine methosulfate (PMS) markedly enhanced cellular reduction of XTT. In the presence of PMS, the XTT reagent yielded usable absorbance values for growth and drug sensitivity evaluations with a variety of cell lines. Depending on the metabolic reductive capacity of a given cell line, the optimal conditions for a 4-h XTT incubation assay were 50 micrograms of XTT and 0.15 to 0.4 microgram of PMS per well. Drug profiles obtained with representative human tumor cell lines for several standard compounds utilizing the XTT-PMS methodology were similar to the profiles obtained with MTT. Addition of PMS appeared to have little effect on the metabolism of MTT. The new XTT reagent thus provides for a simplified, in vitro cell growth assay with possible applicability to a variety of problems in cellular pharmacology and biology. However, the MTA using the XTT reagent still shares many of the limitations and potential pitfalls of MTT or other tetrazolium-based assays.

For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.