Impact of mutant ß-catenin on ABCB1 expression and therapy response in colon cancer cells.

BACKGROUND: Colorectal cancers are often chemoresistant toward antitumour drugs that are substrates for ABCB1-mediated multidrug resistance (MDR). Activation of the Wnt/ß-catenin pathway is frequently observed in colorectal cancers. This study investigates the impact of activated, gain-of-function ß-catenin on the chemoresistant phenotype. METHODS: The effect of mutant (mut) ß-catenin on ABCB1 expression and promoter activity was examined using HCT116 human colon cancer cells and isogenic sublines harbouring gain-of-function or wild-type ß-catenin, and patients' tumours. Chemosensitivity towards 24 anticancer drugs was determined by high throughput screening. RESULTS: Cell lines with mut ß-catenin showed high ABCB1 promoter activity and expression. Transfection and siRNA studies demonstrated a dominant role for the mutant allele in activating ABCB1 expression. Patients' primary colon cancer tumours shown to express the same mut ß-catenin allele also expressed high ABCB1 levels. However, cell line chemosensitivities towards 24 MDR-related and non-related antitumour drugs did not differ despite different ß-catenin genotypes. CONCLUSION: Although ABCB1 is dominantly regulated by mut ß-catenin, this did not lead to drug resistance in the isogenic cell line model studied. In patient samples, the same ß-catenin mutation was detected. The functional significance of the mutation for predicting patients' therapy response or for individualisation of chemotherapy regimens remains to be established.

CHEK2 genomic and proteomic analyses reveal genetic inactivation or endogenous activation across the 60 cell lines of the US National Cancer Institute.

CHEK2 encodes a serine/threonine kinase (Chk2) activated by ATM in response to DNA double-strand breaks. On the one hand, CHEK2 has been described as a tumor suppressor with proapoptotic, cell-cycle checkpoint and mitotic functions. On the other hand, Chk2 is also commonly activated (phosphorylated at T68) in cancers and precancerous lesions. Here, we report an extensive characterization of CHEK2 across the panel of 60 established cancer cell lines from the NCI Anticancer Screen (the NCI-60) using genomic and proteomic analyses, including exon-specific mRNA expression, DNA copy-number variation (CNV) by aCGH, exome sequencing, as well as western blot analyses for total and activated (pT68-Chk2) Chk2. We show that the high heterogeneity of Chk2 levels in cancer cells is primarily due to its inactivation (owing to low gene expression, alternative splicing, point mutations, copy-number alterations and premature truncation) or reduction of protein levels. Moreover, we observe that a significant percentage of cancer cells (12% of the NCI-60 and HeLa cells) show high endogenous Chk2 activation, which is always associated with p53 inactivation, and which is accompanied by downregulation of the Fanconi anemia and homologous recombination pathways. We also report the presence of activated Chk2 (pT68-Chk2) along with histone γ-H2AX in centrosomes.

Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells.

Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

Transcriptomic response to differentiation induction.

BACKGROUND: Microarrays used for gene expression studies yield large amounts of data. The processing of such data typically leads to lists of differentially-regulated genes. A common terminal data analysis step is to map pathways of potentially interrelated genes. METHODS: We applied a transcriptomics analysis tool to elucidate the underlying pathways of leukocyte maturation at the genomic level in an established cellular model of leukemia by examining time-course data in two subclones of U-937 cells. Leukemias such as Acute Promyelocytic Leukemia (APL) are characterized by a block in the hematopoietic stem cell maturation program at a point when expansion of clones which should be destined to mature into terminally-differentiated effector cells get locked into endless proliferation with few cells reaching maturation. Treatment with retinoic acid, depending on the precise genomic abnormality, often releases the responsible promyelocytes from this blockade but clinically can yield adverse sequellae in terms of potentially lethal side effects, referred to as retinoic acid syndrome. RESULTS: Briefly, the list of genes for temporal patterns of expression was pasted into the ABCC GRID Promoter TFSite Comparison Page website tool and the outputs for each pattern were examined for possible coordinated regulation by shared regelems (regulatory elements). We found it informative to use this novel web tool for identifying, on a genomic scale, genes regulated by drug treatment. CONCLUSION: Improvement is needed in understanding the nature of the mutations responsible for controlling the maturation process and how these genes regulate downstream effects if there is to be better targeting of chemical interventions. Expanded implementation of the techniques and results reported here may better direct future efforts to improve treatment for diseases not restricted to APL.

Increased expression of beta 2-microglobulin in multidrug-resistant tumour cells.

The rat monoclonal antibody LMR-12 was shown earlier to react with a plasma membrane protein, upregulated in multidrug-resistant cell lines. In this study, we observed distinct LMR-12 staining in 36 out of 55 non-drug-selected tumour cell lines, including melanomas, renal cell-, colon- and lung carcinomas, whereas in other tumour types, such as leukaemia and ovarian cancer, LMR-12 staining was generally low or absent. The cDNA encoding the LMR-12 antigen was isolated from a library of the multidrug-resistant human fibrosarcoma cell line HT1080/DR4 by expression cloning in MOP8 cells. Sequence analysis showed that the LMR-12 antigen is identical to the major histocompatibility complex class I molecule beta 2-microglobulin (beta2-m). The LMR-12/ beta2-m staining results were confirmed by mRNA microarray data from an independent National Cancer Institute study, as well as by newly obtained reverse transcriptase polymerase chain reaction data. Further analysis of the microarray data showed that beta2-m levels closely reflected levels of major histocompatibility complex class I heavy chains and the transporter associated with antigen processing. Since the ABC transporter associated with antigen processing was previously shown to contribute to multidrug-resistance, it may very well be that the observed LMR-12/ beta2-m levels are secondary to (elevated) levels of the transporter associated with antigen processing. A perspective arising from the present study is that drug resistant tumour cells may, by having elevated levels of major histocompatibility complex related molecules, be particular good candidates for alternative therapeutic therapies, such as cytotoxic T cell mediated immune-therapies.

Characterization of anticancer agents by their growth inhibitory activity and relationships to mechanism of action and structure.

An analysis of the growth inhibitory potency of 122 anticancer agents available from the National Cancer Institute anticancer drug screen is presented. Methods of singular value decomposition (SVD) were applied to determine the matrix of distances between all compounds. These SVD-derived dissimilarity distances were used to cluster compounds that exhibit similar tumor growth inhibitory activity patterns against 60 human cancer cell lines. Cluster analysis divides the 122 standard agents into 25 statistically distinct groups. The first eight groups include structurally diverse compounds with reactive functionalities that act as DNA-damaging agents while the remaining 17 groups include compounds that inhibit nucleic acid biosynthesis and mitosis. Examination of the average activity patterns across the 60 tumor cell lines reveals unique 'fingerprints' associated with each group. A diverse set of structural features are observed for compounds within these groups, with frequent occurrences of strong within-group structural similarities. Clustering of cell types by their response to the 122 anticancer agents divides the 60 cell types into 21 groups. The strongest within-panel groupings were found for the renal, leukemia and ovarian cell panels. These results contribute to the basis for comparisons between log(GI(50)) screening patterns of the 122 anticancer agents and additional tested compounds.

Mdr1 promoter-driven tumor necrosis factor-alpha expression for a chemotherapy-controllable combined in vivo gene therapy and chemotherapy of tumors.

Cancer gene therapy approaches are often designed as single-agent treatments; however, greater therapeutic effect might be obtained if combined with an established conventional treatment regimen such as chemotherapy. In this context, conditional promoters are useful tools, because they may be induced by therapeutic modalities. The human multidrug resistance gene (mdr1) promoter is inducible by cytostatic drugs and can be employed for the chemotherapy-regulated expression of therapeutic genes. In this in vivo study, the human mdr1 promoter fragment (-207 to +158) was used for drug-inducible expression of human tumor necrosis factor-alpha (TNF-alpha) in the vector construct pM3mdr-p-hTNF. The single doxorubicin and vincristine treatment of nude mice xenografted with pM3mdr-p-hTNF-transduced MCF-7 mammary tumors resulted in drug-induced and time-dependent elevation of intratumoral TNF-alpha expression at the mRNA and protein level. The highest drug induction was achieved at 2 days after drug application, as reflected by a maximum 25-fold increase in TNF-alpha secretion in the tumor. This drug-induced TNF-alpha expression is more effective in inhibiting tumor growth compared with the growth of tumors transduced with constitutively TNF-alpha-expressing vectors in combination with chemotherapy.

Novel cytotoxic diterpenes from Casearia arborea.

Cytotoxicity-guided fractionation of the dichloromethane-methanol extract of the roots of Casearia arborea yielded five novel clerodane diterpenes, casearborins A-E (1-5), as well as cucurbitacin B. The presence of cucurbitacins glycosides was also detected. The absolute configuration of casearborin E was determined by X-ray crystallography.

Cytotoxic clerodane diterpene esters from Laetia corymbulosa.

Three cytotoxic clerodane diterpene esters, corymbulosins A-C, were isolated from an organic extract of the fruit of Laetia corymbulosa (Flacourtiaceae) from Peru. The structures were determined by spectroscopic methods as clerodane diterpenes unsaturated at C-3, C-13(16) and C-14. Corymbulosin A was esterified at C-2 with a decadienoate moiety, while corymbulosins B and C were C-2 epimers esterified at C-6 with a decanoate moiety.

Chemotherapy-inducible vector for gene therapy of cancer.

A great variety of viral and nonviral expression systems has been developed and assessed for their ability to transfer genes into somatic cells. In particular, retroviral and adenoviral mediated gene transfer has been extensively studied and improved at least because of their capability to efficiently infect the targeted cells. However, the lack of cell type specificity of viral and nonviral vectors still represents one major obstacle for appropriate and controlled expression of foreign genes. Many attempts have been made to achieve efficient gene delivery by targeting, e.g., retroviral integration via modifications in viral envelope protein sequences, using antibodies as specific mediators in viral infection and pseudotyped viruses, and so on (1, 2, 3, 4).

Cytotoxic geranyl stilbenes from Macaranga schweinfurthii.

Three novel geranyl stilbenes, schweinfurthins A, B, and C (1, 2, and 3), were isolated from the Cameroonian plant Macaranga schweinfurthii (Euphorbiaceae) and their structures determined by NMR and mass spectral methods. The cytotoxicity profile of the schweinfurthins tested in the NCI 60-cell screen was similar to that of the stelletins and cephalostatins, suggesting that these structurally diverse natural products may share similar mechanisms of cytotoxicity.

Recombinant production of cyanovirin-N, a potent human immunodeficiency virus-inactivating protein derived from a cultured cyanobacterium.

Here we describe the recombinant production and purification of a novel anti-human immunodeficiency virus (HIV) protein, cyanovirin-N (CV-N), in Escherichia coli. Initial attempts to express CV-N using a vector containing an ompA signal peptide sequence resulted in production of an intractable mixture of the full-length (101 amino acid residue) protein and a truncated form lacking the first two N-terminal amino acids. The truncated protein was observed regardless of the host cell line, culture conditions, or induction time. These observations suggested that an as yet unidentified protease or peptidase was responsible for proteolytic cleavage between the second and third N-terminal amino acids of CV-N when presented as an ompA-CV-N fusion protein. When the ompA signal peptide sequence was replaced by a pelB signal peptide sequence, CV-N was produced in high yield as a single, homogeneous protein. This was confirmed by electrospray ionization mass spectrometry and N-terminal sequencing. This expression system provides a basis for large-scale production of clinical grade CV-N for further research and development as an anti-HIV microbicide.

Isolation of a novel Kunitz family protease inhibitor in association with Tethya hemolysin from the sponge Tethya ingalli.

Aqueous extracts from the New Zealand sponge Tethya ingalli (Hadromerida) displayed potent cytotoxicity in the NCI's 60-cell-line human tumor panel. Fractionation of the extract by ammonium sulfate precipitation, gel filtration, ultrafiltration, and both hydrophobic interaction and reversed-phase chromatography resulted in the isolation of two biologically active proteins. The first protein, Tethya protease inhibitor (TPI), which was purified to homogeneity, inhibited trypsin with an EC50 of 65 nM. TPI had a molecular mass of 11,431 Da, and an isoelectric point of 8.2. A partial N-terminal amino acid sequence determined for TPI showed significant homology with protease inhibitors of the Kunitz family. The second isolated protein displayed potent cytotoxicity, with pronounced selectivity for certain tumor cell lines (e.g., ovarian, renal, CNS, and breast). The latter protein, which had an apparent molecular weight of 21 kDa (SDS-PAGE), also lysed human red blood cells (EC50 of 39 nM) and was similar to a hemolysin previously isolated from the sponge Tethya lycinurium.

Construction and enhanced cytotoxicity of a [cyanovirin-N]-[Pseudomonas exotoxin] conjugate against human immunodeficiency virus-infected cells.

Cyanovirin-N (CV-N) is a novel 11-kDa anti-HIV(human immunodeficiency virus) protein that binds with high affinity to the viral envelope glycoprotein gp120. In contrast to soluble CD4 and most known neutralizing antibodies that bind gp120, CV-N exerts potent anti-viral activity against primary clinical HIV isolates as well as laboratory-adapted strains of HIV. Here we describe the recombinant production, purification, and characterization of a chimeric toxin molecule, FLAG-CV-N-PE38, that contains CV-N as a gp120-targeting moiety linked to the translocation and cytotoxic domains of Pseudomonas exotoxin A. FLAG-CV-N-PE38 showed enhanced cytotoxicity to HIV-infected, gp120-expressing H9 cells compared to uninfected H9 cells. Competition experiments with free CV-N provided further support that the enhanced FLAG-CV-N-PE38-induced cytotoxicity was due to interactions of the CV-N moiety with cell surface gp120. This study establishes the feasibility of use of CV-N as a gp120-targeting sequence for construction and experimental therapeutic investigations of unique new chimeric toxins designed to selectively destroy HIV-infected host cells.

Increased LRP mRNA expression is associated with the MDR phenotype in intrinsically resistant human cancer cell lines.

Multidrug resistance (MDR) in human cancer cells is multifactorial. Previously, we reported on the association between expression of P-glycoprotein (Pgp), the multidrug resistance-associated protein (MRP), and the lung resistance protein (LRP) with the MDR phenotype in the NCI panel of 60 human cancer cell lines used for in vitro anticancer drug screening. Eight cell lines from this panel, manifesting widely divergent levels of in vitro drug resistance were chosen to investigate the role of MRP and LRP expression at the molecular level. LRP mRNA levels, as determined by ribonuclease protection assay, varied significantly among the 8 cell lines, and correlated closely with in vitro drug resistance to both MDR and non-MDR related drugs. LRP mRNA expression was determined to be a stronger correlate of drug sensitivity than protein expression. In contrast, MRP mRNA levels were not significantly correlated with drug sensitivity. The rates of newly transcribed LRP or MRP mRNA did not correlate with mRNA levels, indicating that mRNA stability or other features of processing may be important in regulation of LRP and MRP mRNA levels. Using Southern blot analysis, LRP gene amplification was shown not to be associated with LRP overexpression. These data suggest that LRP expression may be an important determinant of the MDR phenotype in cell lines intrinsically resistant to cancer chemotherapeutic agents.

Analysis of sequence requirements for biological activity of cyanovirin-N, a potent HIV (human immunodeficiency virus)-inactivating protein.

Site-directed mutagenesis of DNA constructs coding for the novel, HIV-inactivating proteins cyanovirin-N (CV-N) and FLAG-cyanovirin-N (F-CV-N) was performed using mutagenic oligonucleotide primers in the polymerase chain reaction or by a restriction site elimination maneuver. The mutant constructs were expressed in Escherichia coli and the recombinant protein products were tested for binding to the HIV surface envelope glycoprotein gp 120 and for antiviral activity against infectious HIV. Results showed an overall very high correlation (r2 > 0.9) between the relative gp120 binding affinities and the anti-HIV activities of CV-N, F-CV-N, and the various mutants. An outlier, however, was a mutant which lacked one of the internal disulfide linkages normally present in CV-N and which showed modest gp120 binding but no antiviral activity against HIV. These findings are consistent with the view that gp120 binding is a necessary but not sufficient requirement for the HIV-inactivating activity of CV-N and related proteins; the sequence specificities for gp120 binding and anti-HIV activity are not identical.