Heterogeneity within and between primary colorectal carcinomas and matched metastases as revealed by analysis of Ki-ras and p53 mutations

Analysis of the genetic status of Ki-ras and p53 in primary colorectal carcinomas and matched colorectal liver metastasis from 30 patients reveals an overall heterogeneity both within and between the two tumoral tissues. Both genes were found mutated with a similar frequency in both tissues; however, identical mutations in primary tumor and matched metastasis were found less frequently in the case of the Ki-ras than the p53 gene. Only in three cases the same p53 and Ki-ras mutations found in the primary tumor were found also in the metastasis. In several metastatic specimens the DNA bearing a mutation detected also in the primary tumor appears significantly less abundant than the wild-type …

Characterization of transfected HT-29 cells expressing the oncogenic Ras isoform KrasG13D.

Point mutations in codon 12 and 13 of K-ras are frequently found in DNA of colorectal cancer. It has been suggested that particular mutations at these sites may be associated with specific tumour phenotypes. To shed light on the molecular mechanisms on which depends this specificity we set up a system of HT-29 cells stably transfected with a cDNA coding for K-rasG13D under the control of an inducible promoter. Proliferation assay performed on one of the positives clones, showed a decreased growth rate in response to K-rasG13D expression and preliminary gene expression analysis showed an up-regulation of the cell-cycle inhibitor p21 WAF1.

Membrane vesicles containing matrix metalloproteinase-9 and fibroblast growth factor-2 are released into the extracellular space from mouse mesoangioblast Sstem cells

Certain proteins, including fibroblast growth factor-2 (FGF-2) and matrix metalloproteinase-9 (MMP-9), have proved very effective in increasing the efficacy of mesoangioblast stem cell therapy in repairing damaged tissue. We provide the first evidence that mouse mesoangioblast stem cells release FGF-2 and MMP-9 in their active form through the production of membrane vesicles. These vesicles are produced and turned over continuously, but are stable for some time in the extracellular milieu. Mesoangioblasts shed membrane vesicles even under oxygen tensions that are lower than those typically used for cell culture and more like those of mouse tissues. These findings suggest that mesoangioblast…

Specific codon 13 K-ras mutations are predictive of clinical outcome in colorectal cancer patients, whereas codon 12 K-ras mutations are associated with mucinous histotype

Background: K-ras mutations, one of the earliest events observed in colorectal carcinogenesis, are mostly found in codons 12 and 13, and less frequently in codon 61, all three of which are estimated to be critical for the biological activity of the protein. Nevertheless the prognostic significance of such mutations remains controversial. Our purpose was to assess whether any or specific K-ras mutations in primary colorectal cancer had prognostic significance and were linked to clinico-pathological parameters. Patients and methods: Paired tumor and normal tissue samples from a consecutive series of 160 untreated patients (median of follow up 71 months), undergoing resective surgery for prima…

Differential Gene Expression During Corneal Wound Healing

Effetti dell'espressione di K-RasG12V e K-RasG13D in cellule di adenocarcinoma colorettale HT29

RAS è una piccola proteina di 21kDa che si trova frequentemente mutata nei tumori. La famiglia dei geni ras consta di tre principali protoncogeni chiamati H-, K- e N-Ras. Le tre isoforme di Ras regolano la proliferazione, il differenziamento e la morte cellulare mediante l’attivazione di diversi pathways di trasduzione del segnale fra cui la cascata delle MAP chinasi e il pathway di PI3K/AKT. Le diverse isoforme di Ras attivano tutte gli stessi pathways, ma con diversa efficienza, e ciò potrebbe essere, almeno in parte, una conseguenza delle loro differenti modifiche post-traduzionali che determinano la localizzazione in specifici microdomini della membrana plasmatica. Diversi studi hanno d…

Effects of different ras mutations on colorectal cancer cells

Regulation ofMUC1Expression in Human Mammary Cell Lines by the c-ErbB2 and Ras Signaling Pathways

The MUC1 protein is a highly O-glycosylated transmembrane molecule that is expressed at the luminal surface of most glandular epithelial cells and is upregulated in carcinomas. Here, we report the effect of the activation of the c-ErbB2 --Ras pathway on the expression of the MUC1 gene in the nontumorigenic mammary cell lines MTSV1-7 and HB2 and in the malignant cell lines T47D and ZR75. Endogenous levels of MUC1 mRNA and protein in HB2 clones permanently overexpressing c-ErbB2 or V12-H-Ras were markedly reduced compared with levels in the parental cell lines. Furthermore, in transient transfection assays, the transcription of a CAT reporter construct driven by the MUC1 promoter was inhibite…

Caratterizzazione dell'antigene AMA1 in ceppi di Babesia bigemina isolati in Italia

Babesia bigemina is an endemic parasite in different parts of the world, including Europe and the Americas. One of the few genes characterized in this species codifies for the apical membrane antigen 1 (AMA-1), a transmembrane antigen recently identified. We characterized the ama-1 gene from three Italian B. bigemina strains. Italian sequences were compared to those of the Australian strain. The results obtained confirmed that this newly described ama-1 gene is highly conserved among Italian and foreign strains, which has implications for vaccine development.

Lipocalin-2 up-regulation in patients with dry eye. Congresso ARVO 2006.

Intrachromosomal recombination of the c-myc locus leading to gene amplification

Codon 12 and codon 13 mutations in K-RAS differentially affect therapies response of colorectal carcinoma cells

Possible relation between genetic recombination and amplification within the c-myc locus in a case of primary colorectal carcinoma

Genotyping of sicilian Ophrys (Orchidaceae) from Parco delle Madonie

Introduzione a Biologia dello Sviluppo. Principi generali dello Sviluppo.

Con il termine “sviluppo” ci riferiamo fondamentalmente all’insieme degli eventi attraverso cui da un uovo fecondato si genera un organismo pluricellulare, composto da differenti tipi cellulari, organizzati in tessuti ed organi, ciascuno deputato a svolgere una specifica funzione (di sostegno, di protezione, di nutrizione, di locomozione, sensoriale, riproduttiva, ecc.) e con una sua ben precisa forma tridimensionale e localizzazione spaziale rispetto alle altre strutture che compongono l’organismo.

Lipocalin-2-up regulation in patients with dry eye

Gene expression profiling of HT-29 cells in response to induction of oncogenic H and K-ras.

Studies on sea urchin oocytes. II. Synthesis of RNA during oogenesis.

Abstract Isolated oocytes of the sea urchin Paracentrotus lividus actively incorporate 3H-uridine into RNA. Labeled RNA was analysed by sucrose gradient and acrylamide gel electrophoresis following cell fractionation. Much of the radioactivity is incorporated at the nucleolar level in the form of rRNA precursors. The kinetics of maturation of these latter suggests that this occurs at a slower rate than during embryogenesis. Other non-nucleolar RNA classes are also actively labelled and retained in the nucleus for many hours. These results are confirmed by an autoradiographic investigation.

DUE DIFFERENTI MECCANISMI MOLECOLARI COINVOLTI NEL FENOMENO DELL'AMPLIFICAZIONE GENICA IN UN CASO DI CARCINOMA COLORETTALE

Havep53 gene mutations and protein expression a different biological significance in colorectal cancer?

p53 alterations are considered the most common genetic events in many types of neoplasms, including colorectal carcinoma (CRC). These alterations include mutations of the gene and/or overexpression of the protein. The aim of our study was to assess whether in 160 patients undergoing resective surgery for primary operable CRC there was an association between p53 mutations and protein over-expression and between these and other biological variables, such as cell DNA content (DNA-ploidy) and S-phase fraction (SPF), and the traditional clinicopathological variables. p53 mutations, identified by PCR-SSCP-sequencing analysis, were found in 68/160 patients (43%) and positive staining for p53 prote…

Analisi comparativa dei livelli di RNA in cellule in coltura trattate con dexametasone

Intrachromosomal recombination of c-myc locus leading to gene amplification

Electrophoretic separation of a class of nucleosomes enriched in HMG 14 and 17 and actively transcribed globin genes.

Monomer nucleosomes from chick erythrocytes can be fractionated according to their electrophoretic mobility in (comparatively) high salt acrylamide gels. We show that the fractionation is based predominantly on differences in charge. The monomer heterogeneity persists even when the nucleosomes are trimmed down to 145 bp with Exo III or when H1 and H5 are removed. The slowest migrating monomers are associated with HMG 14 and 17; however, we do not believe that these proteins are entirely responsible for the altered mobility since the nucleosome heterogeneity persists even after removal of HMG 14 and 17. The DNA associated with the HMG 14 and 17 containing nucleosomes is shown to be enriched …

Differential effects of oncogenic H- and K-ras expression on HT-29 colorectal carcinoma cell line.

The Ras oncogene is mutated in about 30% of the human tumors and its mutations are always point mutations concerning codon 12, 13 and 61. These mutations cause in the proteins a reduced GTPase activity, so that they become constitutively active. In human cells there are three main isoforms of Ras (H, K, N-ras) which can trigger alternative pathways of signal trasduction. In order to investigate the effects of expression of different oncogenic Ras isoforms in colorectal carcinoma cells (HT-29), we obtained stable clones of HT-29 cells transfected with cDNAs codifying H-RasG12V and K-Ras G12V called respectively H12 and K12 and K-RasG13D called K13, under the control of an hormone-inducible p…

Analisi degli effetti del Cetuximab in linee cellulari di adenocarcinoma colorettale

Il carcinoma del colon-retto (CRC) è una malattia eterogenea, che si sviluppa in seguito a numerose alterazioni genetiche ed epigenetiche. Una famiglia genica che si trova frequentemente mutata nei tumori è quella di ras che consta di tre principali protoncogeni (H-, K- e N-Ras) localizzati su cromosomi differenti e codificanti proteine G del peso di 21 KDa. Il 50% dei casi di CRC presenta mutazioni puntiformi missense a carico del gene KRAS, il 90% delle quali si verifica a livello dei codoni 12 e 13 e rende la proteina costitutivamente attiva. Diversi studi dimostrano come mutazioni di differenti codoni di K-Ras potrebbero avere diverse conseguenze biologiche e determinare una diversa ris…

Anfibi

Effescts of different RAS mutations on colorectal cancer cells

DNA Extraction From Orthoptera Museum Specimens

We describe a procedure for rapid purification of high quality DNA from either fresh or dry Orthoptera, suitable for the PCR amplification of DNA regions more than 800 bp long (even from oldest specimens), which allows genetic analyses on animals from collections without the complete specimen disruption.

Two distinct amplification events of the c-myc locus in a colorectal tumour.

Southern hybridisation of genomic DNA extracted from a human primary colorectal carcinoma revealed amplification of a fragment containing the wild-type c-myc locus. Two additional rearranged DNA fragments, lying upstream of c-myc, fused to distant non-contiguous sequences from the same chromosome, with an opposite configuration (head to head vs. head to tail), were also found to be amplified. Sequences analysis suggested that these rearrangements resulted from illegitimate recombination at two distinct points within the DNA sequence just upstream of the c-myc ORF and further that these events triggered two different amplification mechanisms, only one of which, involving a strand invasion ev…

Characterization of the Apical Membrane Antigen-1 in Italian Strains of Babesia bigemina

Summary Babesia bigemina is a parasite endemic in different parts of the world, including Europe and the Americas. One of the few genes characterized in this species codifies for the Apical Membrane Antigen 1 (AMA-1), a trans-membrane antigen recently identified. In this research, we characterized the ama-1 gene from three Italian B. bigemina strains, two B. bigemina strains obtained from Ragusa, Sicily (ITA1 and ITA3) and a third one obtained from Benevento, Campania (ITA2). Italian sequences were compared with those of the Australian strain obtained from the Sanger Institute web site and to strains from different parts of the world. The results obtained confirmed that this newly described…

Babesia bigemina Apical Membrane Antigen-1: features of the gene and predictive information of the protein from Italian strains

Babesia bigemina is a tick-borne haemoparasite affecting cattle in different parts of the world. One of the few antigens characterized in this species codifies for the Apical Membrane Antigen 1 (AMA-1), a transmembrane protein recently identified. In this work, we characterized the ama-1 gene from four Italian B.bigemina strains, two obtained from Ragusa, Sicily (ITA1 and ITA3), one obtained from Benevento, Campania (ITA2) and one coming from Perugia, Umbria (ITA4). Italian sequences were compared to the Australian strain, whose sequence is available in the Sanger Institute web site, and to some strains coming from different parts of the world. This study provides useful information on the …

Role of H-RAS and K-RAS oncogenes on mucin expression in colorectal carcinoma

Analysis of the mechanisms through which K-RASG12V and K-RASG13D regulate the proliferation and cell death in cells HT-29

Different micrococcal nuclease cleavage patterns characterize transcriptionally active and inactive sea-urchin histone genes.

Micrococcal nuclease (MNase) and DNaseI have made a great contribution to our present understanding of the structural organization of the eucaryotic genome [l - 31. The enhanced sensitivity of active portions of the genome to DNaseI gave, in fact, the first indication of differences in the nucleoproteic arrangement of the transcribed as compared to the silent DNA regions [4]. The use of MNase, as a probe of the chromatin organization of specific genes in the active and inactive state, provided additional evidence for this and further showed that the packing of several coding segments of DNA in a regular array of nucleosomal particles is severely but reversibly affected by the transcriptiona…

Lipocalin-2 up-regulation in patients with dry eye.

Codon 12 and codon 13 mutations in K-RAS differentially affect colorectal carcinoma cells

AMPLIFICAZIONE E RIARRANGIAMENTO NEI TUMORI COLORETTALI

Havep53gene mutations and protein expression a different biological significance in colorectal cancer?*

p53 alterations are considered the most common genetic events in many types of neoplasms, including colorectal carcinoma (CRC). These alterations include mutations of the gene and/or overexpression of the protein. The aim of our study was to assess whether in 160 patients undergoing resective surgery for primary operable CRC there was an association between p53 mutations and protein overexpression and between these and other biological variables, such as cell DNA content (DNA-ploidy) and S-phase fraction (SPF), and the traditional clinicopathological variables. p53 mutations, identified by PCR-SSCP-sequencing analysis, were found in 68/160 patients (43%) and positive staining for p53 protei…

Direct Identification of Each Specific Mutation in Codon 12 and 13 of ci-ki-ras2 by SSCP Analysis

We compared the SSCP behaviour of the DNA fragments containing c-ki-ras 2 wild type 12 and 13 codons or each of the 12 possible point mutated sequences in these two codons. We found that a single electrophoresis condition was sufficient to distinguish each specific mutation from the other 11 and from the wild type sequence. This observation makes it possible to identify each specific mutation directly by SSCP without any need for reamplification and sequencing.

Analysis of the effects of K-RasG12V and K-RasG13D on the cell cycle

p21 Ras is small protein with GTPase activity that regulates proliferation, differentiation and apoptosis in all cell types. The three major isoforms of Ras (H-, K- and N-Ras) differing only for the last 24 aminoacids have different post-translational modifications that lead to localization in diverse plasma membrane microdomains and downstream activation of alternative pathways of signal transduction. This might explain, at least in part, the different biological effects of the Ras isoforms in the cells. Ras mutations are a common event in several tumours and in almost all cases they are point mutations in codons 12 or 13, and rarely in codon 61. These mutations lead to a constitutively ac…