A mutational analysis of phosphatidylinositol-3-kinase activation by human colony-stimulating factor-1 receptor.

Colony-stimulating factor-1 (CSF1) is a cell lineage-specific hemopoietin required for the growth, differentiation, and survival of macrophages and their precursors. The human CSF1 receptor (CSF1R) is a 150-kDa transmembrane glycoprotein whose cytoplasmic tyrosine kinase domain is split by a kinase insert (KI) region of approximately 70 amino acids. We tested the ability of CSF1R KI domain deletion mutants to stimulate phosphatidylinositol-3-kinase (PtdIns-3-kinase), an enzyme whose activity is augmented by tyrosine kinase oncogenes and receptor tyrosine kinases, and to support mitogenesis in transfected cells. Receptor immunoprecipitates from CSF1-stimulated cells contained greater than 5-fold more PtdIns-3-kinase activity compared to nonstimulated cells. High performance liquid chromatography analysis of the PtdIns-3-kinase product scraped from thin layer chromatography plates indicated that PtdIns-3-P was produced. CSF1R KI domain deletion mutants retained tyrosine kinase activity in vitro. Receptor immunoprecipitates of two partially overlapping 28 and 30 amino acid KI deletion mutants of CSF1R retained some PtdIns-3-kinase activity, in contrast to immunoprecipitates of CSF1R lacking 67 amino acids of the KI domain. Each deletion mutant stimulated CSF1-dependent DNA synthesis in transfected cells at much reduced levels compared to wild-type receptor expressing cells. These data suggest a role for the CSF1R KI domain in PtdIns-3-kinase association and for CSF1-induced thymidine incorporation into DNA.     

Characterization of a novel oncogenic K-ras mutation in colon cancer

Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity.     

A point mutational analysis of human papillomavirus type 16 E7 protein.

The E7 open reading frame of human papillomavirus type 16 (HPV16) has been shown to be selectively retained in cervical tumors and to encode both transforming and trans-activating functions in murine cells, supporting the notion that expression of E7 contributes towards the progression of premalignant cervical lesions. A comparison among E7 sequences of different HPV types reveals some homology at the amino acid level. Of particular interest are two regions, one which contains significant homology to a region of adenovirus E1a and simian virus 40 large T (LT), and a second region which contains two conserved Cys-X-X-Cys motifs. To determine the importance of these domains to the function of the E7 protein, a series of mutants carrying substitutions at amino acids in the region of E1a-LT homology and at the Cys-X-X-Cys motifs were constructed. The mutated E7 sequences were placed under the control of a strong heterologous promoter (Moloney long terminal repeat), and the activity of the mutants was assayed in NIH 3T3 cells, a cell line in which both the transforming function and the trans-activating function of E7 could be determined. A single amino acid substitution analogous to a mutation in E1a which destroys the transforming ability of this protein abolished both transformation and trans-activation by E7. Mutations at the Cys-X-X-Cys motifs demonstrated that this region contributes to the transforming potential of E7, although proteins in which both motifs were interrupted retained a low level of transforming activity. Mutations in the region of E1a-LT homology which occur within a recognition sequence for casein kinase II did not markedly affect transforming activity of E7 but severely reduced trans-activating ability. This indicates that efficient trans-activation is not required for transformation by HPV16 E7 in these cells.     

Generation of ribozyme-adenoviral vector against K-ras mutant human lung cancer cells

ras mutations represent one of the most common oncogenetic lesions in human non-small cell lung cancer (NSCLC) and adversely affect the survival of patients afflicted with this disease. ras-directed gene therapy in the past employed primarily antisense oligonucleotides (AS-ODN) or expression vectors (such as a viral vector construct) that deliver the antisense sequence to inactivate the mutant oncogene message. These approaches produced minimal toxicity, and yet were limited in efficacy. Ribozymes present a viable alternative in antisense therapy by virtue of their renewable catalytic capability for site-specific RNA cleavage. We recently produced an adenoviral vector with a hammerhead ribozyme transgene (KRbz) that is specific for the K-ras codon 12 mutant sequence GUU, given the considerations that (a) in the United States, approx 30% of human NSCLCs express K-ras oncogene mutations, nearly all of which reside in codon 12; (b) anti-K-ras, anti-H, as well as anti-N-ras hammerhead ribozymes are potent growth inhibitors in various human cancers tested; and (c) in vitro and animal model studies suggest that ribozymes directed at oncogene (K- and H-ras C-fos, BCR-ABL) or human immunodeficiency viral gene messages are more effective than their antisense counterpart. This article describes the techniques involved in the production of the KRbz-adenoviral vector that is specific for the K-ras mutation GTT, and summarizes its in vivo antitumor effect against NSCLC xenografts expressing the relevant K-ras mutation in athymic mice.     

Topography of mutational signatures in human cancer

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Detection of micro-RNA by a combination of nucleic acid sequence-based amplification and a novel chemiluminescent pyrophosphate assay

Micro-RNA has attracted much attention as a biomarker for disease progression and malignancy. A compact, simple, rapid, and highly sensitive method is required to perform simple genetic analyses, such as point-of-care testing (POCT), at the clinic or bedside. Nucleic acid sequence-based amplification (NASBA) is a specific amplification method for a single-stranded RNA fragment that is useful for the highly sensitive detection of miRNAs. In this work, we developed a novel miRNA analytical system for POCT by combining the NASBA and chemiluminescence methods. Because the NASBA reaction is conducted at a constant temperature (41°C) and detection by chemiluminescence reaction does not require a light source, these methods could be combined to amplify 100 ng/assay miRNA. This combined miRNA detection method could be useful for the future development of compact POCT systems.     

A novel activating mutation of the K-ras gene in human primary colon adenocarcinoma

We identified a novel type of point mutation at the 22nd codon of the K-ras gene in a primary colon cancer. The mutation was C to A transversion substituting lysine (AAG) for normal glutamine (CAG) codon. Biological activity of this mutant K-ras gene was tested by expression of full-length cDNA clones in NIH3T3 cells. Most of the K-ras Lys22-transfected cells exhibited an increased saturation density, a lower serum requirement, and transformed morphology reminiscent to the typical K-ras Val12 transformants. However, the tumorigenicity of K-ras Lys22 transformants in nude mice was significantly less potent than that of K-ras Val12 transformants; only a high copy number transformant produced tumors. Even though the activation is incomplete, the finding that the majority of tumor cells in the specimen carried the K-ras Lys22 mutation suggests that this mutation might be advantageous for growth of tumor cells in vivo.     

Pilot study of a novel combination of two therapeutic vaccines in advanced non-small-cell lung cancer patients

Cancer vaccines contain tumor antigens in a pro-inflammatory context with the purpose to generate potent antitumor immune responses. However, tumor cells develop different immunosuppressive mechanisms that limit the effectiveness of an anticancer immune response. Therefore, therapeutic vaccine treatment alone is usually not sufficient to generate tumor regression or survival improvement, especially in the advanced disease scenario in which most clinical studies have been conducted. Combining cancer vaccines with different anticancer therapies such as chemotherapy, radiotherapy and other immunotherapeutic agents has had different levels of success. However, the combination of cancer vaccines with different mechanisms of action has not been explored in clinical trials. To address this issue, the current review summarizes the main clinical and immunological results obtained with two different therapeutic vaccines used in advanced non-small-cell lung cancer patients, inducing an immune response against epidermal growth factor (CIMAvax-EGF) and NGcGM3 ganglioside (racotumomab). We also discuss preliminary findings obtained in a trial of combination of these two vaccines and future challenges with these therapies.     

Increased K-ras protein and activity in mouse and human lung epithelial cells at confluence.

Although K-ras is frequently mutated in lung adenocarcinomas, the normal function of K-ras p21 in lung is not known. In two mouse (E10 and C10) and one human (HPL1D) immortalized lung cell lines from peripheral epithelium, we have measured total K-ras p21 and active K-ras p21-GTP during cell proliferation and at growth arrest caused by confluence. In all three cell types, total K-ras p21 increased 2- to 4-fold at confluence, and active K-ras p21-GTP increased 10- to 200-fold. It was estimated that 0.03% of total K-ras p21 was in the active GTP-bound state at 50% confluence, compared with 1.4% at postconfluence. By contrast, stimulation of proliferation by serum-containing medium did not involve K-ras p21 activation, even though a rapid, marked activation of both Erk1/2 and Akt occurred. At confluence, large increases, up to 14-fold, were seen in Grb2/Sos1 complexes, which may activate K-ras p21. In sum, increased protein expression and activity of K-ras p21 are associated with growth arrest, not with proliferation, in mouse and human lung cell lines.     

Comparison of K-ras gene mutations in tumour and sputum DNA of patients with lung cancer.

Mutations in the K-ras gene are frequently found in lung tumours and are implicated in the development of lung cancer. In order to investigate the clinical usefulness of these mutations in lung cancer, we applied a sensitive method to compare mutations in codon 12 of the K-ras gene in DNA extracted from lung tumours and the matched sputum samples obtained from 22 lung cancer patients. K-ras mutations were identified in the lung tumours of 12 patients (54.5%) and in the sputum samples of 10 patients (45.5%). Nine patients showed an identical mutation in both the tumour and the matched sputum samples. There was a significant association between the presence of a K-ras mutation in a lung tumour and the detection of an identical mutation in the matched sputum sample of the lung cancer patient (kappa = 0.64, 95% confidence interval 0.32-0.95, p <0.01). K-ras mutations were detected in sputum samples from cancer patients with all lung tumour grades, and both in the presence and the absence of lymph node metastasis. Therefore, K-ras mutations may provide useful diagnostic markers for lung cancer.     

A novel mutational hotspot in a natural quasipalindrome in Escherichia coli

We have found that most spontaneous mutations in the thyA gene of Escherichia coli selected for resistance to trimethoprim result from a TA to AT transversion at a single site within an imperfect inverted repeat or quasipalindrome sequence. This natural quasipalindrome within the coding region of thyA contains an extraordinarily potent hotspot for mutation. Our analysis provides evidence that these mutations are templated by nearby sequences by replication within a hairpin structure. Although quasipalindrome-associated mutations have been observed in many organisms, including humans, the cellular avoidance mechanisms for these unusual mutational events have remained unexplored. We find that the mutational hotspot in thyA is dramatically stimulated by inactivation of exonucleases I and VII, which degrade single-strand DNA with a common 3'-5' polarity. We propose that these exonucleases abort the replicative misalignment events that initiate hairpin-templated mutagenesis by degrading displaced nascent DNA strands. Mismatch repair-defective strains also showed increased mutability at the hotspot, consistent with the notion that these mutations arise during chromosomal lagging-strand replication and are often subsequently removed by methyl-directed mismatch repair. The absence of the thyA quasipalindrome sequence from other related bacterial genera suggests that this sequence represents a "selfish" DNA element whose existence itself is driven by this unusual hairpin-templating mechanism.     

Functional changes in a novel uracil-DNA glycosylase determined by mutational analyses

Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in bacteria and eukaryotes, which removes uracil residues from DNA strands. Methanococcus jannaschii UDG (MjUDG), a novel monofunctional glycosylase, contains a helix-hairpin-helix (HhH) motif and a Gly/Pro rich loop (GPD region), which is important for catalytic activity; it shares these features with other glycosylases, such as endonuclease III. First, to examine the role of two conserved amino acid residues (Asp150 and Tyr152) in the HhH-GPD region of MjUDG, mutant MjUDG proteins were constructed, in which Asp150 was replaced with either Glu or Trp (D150E and D150W), and Tyr152 was replaced with either Glu or Asn (Y152E and Y152N). Mutant D150W completely lacked DNA glycosylase activity, whereas D150E displayed reduced activity of about 70% of the wild type value. However, the mutants Y152E and Y152N retained unchanged levels of UDG activity. We also replaced Glu132 in the HhH motif with a lysine residue equivalent to Lys120 in endonuclease III. This mutation converted the enzyme into a bifunctional glycosylase/AP lyase capable of both removing uracil at a glycosylic bond and cleaving the phosphodiester backbone at an AP site. Mutant E132K catalyzes a β-elimination reaction at the AP site via uracil excision and forms a Schiff base intermediate in the form of a protein-DNA complex. This text was submitted by the authors in English.     

Towards a genetic-based classification of human lung cancer.

Lung cancer is a highly aggressive neoplasm which is reflected by a multitude of genetic aberrations being detectable on the chromosomal and molecular level. In order to understand this seemingly genetic chaos, we performed Comparative Genomic Hybridisation (CGH) in a large collective of human lung carcinomas investigating different tumor entities as well as multiple individual tumour specimens of single patients. Despite the considerable genetic instability being reflected by the well known morphological heterogeneity of lung cancer the comparison of different tumour groups using custom made computer software revealed recurrent aberration patterns and highlighted chromosomal imbalances that were significantly associated with morphological histotypes and biological phenotypes. Specifically we identified imbalances in NSCLC being associated with metastasis formation which are typically present in SCLC thus explaining why the latter is such an aggressive neoplasm characterized by widespread tumor dissemination. Based on the genetic data a new model for the development of SCLC is presented. It suggests that SCLC evolving from the same stem cell as NSCLC should be differentiated into primary and secondary tumors. Primary SCLC corresponding to the classical type evolved directly from an epithelial precursor cell. In contrast, secondary SCLC correlating with the combined SCLC develops via an NSCLC intermediate. In addition, we established libraries of differentially expressed genes from different human lung cancer types to identify new candidate genes for several of the chromosomal subregions identified by CGH. In this review, we summarise the status of our results aiming at a refined classification of lung cancer based on the pattern of genetic aberrations.     

Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line.

The c-erbB-2 gene is a v-erbB-related proto-oncogene which is distinct from the gene encoding the epidermal growth factor receptor. By using two independent methods, hybridization of both sorted chromosomes and metaphase spreads with cloned c-erbB-2 DNA, we mapped the c-erbB-2 locus on human chromosome 17 at q21, a specific breakpoint observed in a translocation associated with acute promyelocytic leukemia. Furthermore, we observed amplification and elevated expression of the c-erbB-2 gene in the MKN-7 gastric cancer cell line. These data suggest possible involvement of the c-erbB-2 gene in human cancer.