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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 risposta ai trattamenti farmacologici. Negli ultimi anni sono state sviluppate diverse terapie per il trattamento del CRC, tra i nuovi farmaci vi è il Cetuximab, un anticorpo monoclonale IgG1 chimerico uomo/topo, che agisce contro il dominio extracellulare dell’EGFR (Epidermal Growth Factor Receptor). Il legame di questo anticorpo al recettore comporta una diretta inibizione della sua attività tirosin-chinasica provocando il blocco della trasduzione del segnale mediato da pathways quali quello di PI3K/AKT, e di RAS-RAF/MAPKs, inducendo quindi meccanismi proapoptotici e blocco della proliferazione cellulare. Numerosi trials clinici condotti negli ultimi anni hanno dimostrato che i pazienti con CRC che hanno mutazioni in K-RAS nel codone 12 o 13 rappresentano una popolazione eterogenea nella risposta al trattamento con il Cetuximab e sono attualmente esclusi dal trattamento con tale farmaco. Per valutare come cellule con mutazioni differenti rispondano a trattamenti con Cetuximab, nel nostro laboratorio abbiamo iniziato a condurre esperimenti sulla linea cellulare di CRC HT-29 caratterizzata da mutazioni nei geni BRAF e PIK3CA, e dotata di KRAS wild type, e su due cloni di HT-29 stabilmente trasfettati con i cDNA codificanti per K-RasG12V e K-RasG13D posti sotto il controllo di un promotore inducibile dall’ormone Mifepristone [GeneSwitchTM System (Invitrogen)]. Le diverse linee cellulari sono state sottoposte a trattamento con diverse concentrazioni di Cetuximab ed è stata valutata la proliferazione cellulare dopo 24, 48 e 72 ore di trattamento. Dai primi risultati ottenuti sembra che le cellule con la mutazione K-RASG13D siano parzialmente sensibili al trattamento con Cetuximab mostrando un’alterazione nel tasso di proliferazione cellulare mentre le cellule parentali HT29 e il clone K-RASG12V sembrano non risentire di alcun effetto qualunque sia la concentrazione di farmaco utilizzata. Questi primi risultati seppure da confermare e supportare con ulteriori indagini biomolecolari sembrano avvalorare l’ipotesi secondo cui tumori caratterizzati da mutazioni differenti rispondono in maniera differente alle terapie oggi in uso.

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 dimostrato come l’incidenza di mutazioni dei geni ras varia tra i diversi tipi tumorali ed è stata riscontrata una certa correlazione tra il tipo di tumore e l’isoforma di Ras mutata. K-Ras si trova più frequentemente mutato nel carcinoma colorettale e le alterazioni sono per il 90% mutazioni puntiformi nel codone 12 o 13 e raramente nel codone 61. I residui aminoacidici che corrispondono nella proteina a questi codoni sporgono nel sito catalitico e sono importanti per l’attività GTPasica, quindi mutazioni in questi siti rendendo Ras iperattiva. Molti studi riportano che in differenti tumori primari o metastatici mutazioni di differenti codoni, o differenti mutazioni nello stesso codone e mutazioni in isoforme diverse di Ras potrebbero avere conseguenze biologiche diverse. Per comprendere le basi molecolari dei differenti effetti delle mutazioni di Ras, abbiamo usato cellule HT-29 (una linea cellulare di adenocarcinoma colorettale in cui i geni Ras sono wild-type) stabilmente trasfettate con i cDNA codificanti per K-RasG12V e K-RasG13D posti sotto il controllo di un promotore inducibile dall’ormone Mifepristone. Nel nostro laboratorio abbiamo trovato che l’espressione di queste due forme mutate di Ras causa differenti effetti sul tasso di crescita cellulare. Saggi di citofluorimetria hanno mostrato anche un differente effetto sul ciclo cellulare e che l’espressione di K-RasG13D ma non di K-RasG12V causa la morte cellulare per apoptosi o per necrosi. Entrambe le isoforme mutate di Ras incrementano l’espressione dell’inibitore del ciclo cellulare p21Waf-1/Cip-1. Infine, il trattamento di entrambi i cloni con l’inibitore delle chinasi MEK1/2, il PD98059, e/o con l’inibitore della PI3K, l’LY294002, aumenta l’espressione della proteina p21Waf-1/Cip-1, altera la proliferazione cellulare e la distribuzione delle cellule nelle tre fasi del ciclo cellulare.

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.

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.

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 features of B.bigemina AMA-1 gene from Italian strains of the parasite and some predictive information of the protein obtained by bioinformatics. A comparison among Italian strains of B.bigemina showed a very high degree of both nucleotide and aminoacidic identity. The gene resulted highly conserved among Italian and foreign strains and the protein was included in the AMA-1 family. By the means of bioinformatics it was possible to compare some features of B.bigemina AMA-1 protein with the already characterized Plasmodium falciparum orthologue. Predictive results show that a structural and thus functional homology between B.bigemina and P.falciparum AMA-1 can be supposed. Moreover several putative antigenic peptides were detected and they could be used for investigations about the recognition by specific antibodies. These data put the basis for a further study on B.bigemina AMA-1 antigen.

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.

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 promoter. We had previously observed changes in shape and growth rate when mutated Ras was expressed. We then analyzed the differential expression of genes such as the cell-cycle inhibitor p21 and the EMT associated gene snail. Q-RT PCR assay showed an increase in p21 level only in H12 cells but not in K13 and K12 compared with the respective controls. Instead, snail mRNA level was increased in all thre clones. We investigated also by Semiquantitative RT-PCR H-RasG12V, p21 and snail levels in H12 cells induced for a long time and we noted a decreased level of H-RASG12V associated with no p21 and snail expression. Our results show that specific mutations of different Ras isoforms have different effects on morphology and gene expression in Ht-29. Other studies are of course necessary to shed more light on the role played by Ras mutations in tumor development.

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 protein, detected with the monoclonal antibody DO-7, was present in 48% (77/160) of the cases, with agreement of 57% (91/160). In particular, a significant association was found between increased p53 expression and genetic alterations localized in the conserved regions of the gene or in the L3 DNA-binding domain and the specific type of mutation. Furthermore, both overexpression of p53 and mutations in the conserved areas of the gene were found more frequently in distal than in proximal CRCs, suggesting that they might be "biologically different diseases." Although p53 mutations in conserved areas were associated with flow cytometric variables, overexpression of p53 and mutations in its L3 domain were only related respectively to DNA-aneuploidy and high SPF. These data may reflect the complex involvement of p53 in the different pathways regulating cell-cycle progression. In conclusion, the combination of the mutational status and immunohistochemistry of p53, and flow cytometric data may provide an important insight into the biological features of CRCs.

RAS is a family of small proteins with GTPase activity that regulate proliferation, differentiation and apoptosis in all cell types. The three major isoforms of RAS (H-, K- and N-RAS) differ only in the last 25 amino acids which are the site of different post-translational modifications that lead to diverse subcellular localization and efficiency of 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 tumorigenesis and in colorectal carcinomas the mutations in K-RAS are more frequent than mutations in H-RAS. In almost all cases, the genetic alteration is a point mutation in codons 12 or 13, and rarely in codon 61. These mutations lead to a constitutively active protein by inactivating its GTPase activity. However, data indicate in different primary and metastatic tumors that not only mutations of different isoforms of RAS, but also mutations in different codons or different mutations in the same codon of the same isoform of RAS may have diverse biological consequences. To shed more light on the molecular mechanisms responsible for the different effects of RAS mutations, we have used stable clones of HT-29 (a human colorectal adenocarcinoma cell line in which the endogenous RAS genes are wild type) transfected with cDNAs codifying: K-RASG12V (clone K12) and K-RASG13D (clone K13) under the control of a Mifepristone-inducible promoter. We found that the expression of each of these mutated RAS isoforms induces different effects on the growth rate. Cytofluorimetric analysis shows also a differential effect on the cell cycle. In particular, we observe a significant increase in the fraction of cells in the G2/M phase, more relevant in the K13 than in the K12 induced cells. For a better understanding of this phenomenon we investigated the p21CIP1/WAF1 protein intracellular levels by western blot, identifying a similar increase in the amount of p21 in both clones upon induction of the mutated K-RAS isoforms. This increase persisted, and was actually enhanced, even in the presence of inhibitors of the two main signal transduction pathways operating downstream of RAS, the MEK inhibitor PD98059 and the PI3K inhibitor LY294002. B-RAF and RAF-1 expression is instead decreased by both the mutated isoforms of RAS, in growing as well as in starved cells. The two RAS mutations show the ability to differentially influence the pro-apoptotic (MST2-RASSF1A-LATS1) and anti-apoptotic (MST2-RAF-1) pathways. Cellular death was analyzed by propidium iodide and the caspase assay. The data show an increase in total cell death rate in both induced clones, but K-RASG13D preferentially induced a significant increase in necrosis while K-RASG12V in apoptosis, with a less pronounced effect. The K-RASG13D interactome was also analysed and compared to that of unstimulated wild type K-RAS by mass spectrometry in Hke3 cells in starving condition (0% serum).