In the last years small RNA molecules, i.e. microRNA (miRNA) encoded by miR genes, have been found to play a crucial role in regulating gene expression of a considerable part of plant's and animal's genome. Here, we report the essential information on biogenesis of miRNAs and recent evidence on their important role in human diseases. Emphasis has been given to miR-155, since this molecule represents a typical multifunctional miRNA. Recent data indicate that miR-155 has distinct expression profiles and plays a crucial role in various physiological and pathological processes such as haematopoietic lineage differentiation, immunity, inflammation, cancer, and cardiovascular diseases. Moreover, miR-155 has been found to be implicated in viral infections, particularly in those caused by DNA viruses. The available experimental evidence indicating that miR-155 is over expressed in a variety of malignant tumors allows us to include this miRNA in the list of genes of paramount importance in cancer diagnosis and prognosis. Exogenous molecular control in vivo of miR-155 expression could open up new ways to restrain malignant growth and viral infections, or to attenuate the progression of cardiovascular diseases.

Postreplicative mismatch repair plays a major role in mediating the cytotoxicity of agents generating O6-methylguanine in DNA. We previously showed that a methylating antitumor triazene compound, temozolomide, induces apoptosis and that the persistence of O6-methylguanine in DNA is required to trigger the process. We wanted to test whether the latter apoptotic signal is dependent on a functional mismatch repair system. To this end, we used two human lymphoblastoid cell lines (i.e., the mismatch repair-proficient TK6 line and its mismatch repair-deficient subline MT1) that are both deficient in O6-methylguanine repair. Temozolomide treatment of TK6 cells brought about efficient cell growth inhibition, G2/M arrest, and apoptosis, as indicated by the results of cytofluorimetric analysis of 5-bromo-2'-deoxyuridine incorporation and DNA content and evaluation of DNA fragmentation. The drug treatment resulted also in the induction of p53 and p21/waf-1 protein expression. In contrast, MT1 cells were highly resistant to the drug and no p53 and p21/waf-1 induction was observed. Importantly, we could show that MT1 cells are not deficient in the p53-dependent apoptosis pathway; treatment with etoposide, a topoisomerase II inhibitor, resulted in p53 and p21/waf-1 protein expression and apoptosis in both cell lines. In conclusion, we demonstrate the existence of a link between a functional mismatch repair system and the trigger of apoptosis in cells exposed to clinically relevant concentrations of temozolomide. The results also suggest that p53 induction in response to O6-guanine methylation involves the mismatch repair system.

The results show that our regimen has strong immunologic and antitumor activity in colorectal cancer patients and deserves to be investigated in phase III trials.

No abstract

The prognosis of advanced melanoma is generally poor, because this tumor commonly exhibits intrinsic or acquired resistance to chemotherapy. In an attempt to identify the underlying causes of this resistance, we studied the roles played by the DNA repair enzyme O 6 -alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair (MMR) system in the sensitivity of melanoma cells to temozolomide (TMZ), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), or cis-diamminedichloroplatinum(II) (CDDP). To this end, OGAT levels and MMR efficiency of extracts of nine melanoma cell lines and selected clones derived from four of these lines were determined and correlated with the sensitivity of the respective cells to these drugs. The effectiveness of O 6 -benzylguanine (BG), a specific OGAT inhibitor, in potentiating TMZ-or BCNU-mediated cytotoxicity was also evaluated. Our results demonstrate that MMR efficiency and OGAT levels strongly affect melanoma cell sensitivity to TMZ. In MMR-proficient cells, a direct correlation between OGAT levels and TMZ IC 50 values was found. When OGAT activity was inhibited with BG, the sensitivity of these cells to TMZ increased and was then dictated largely by their MMR efficiency. MMR-deficient cells were highly resistant to the drug irrespective of their OGAT levels. Although OGAT activity and MMR status seemed to be the major determinants of melanoma sensitivity to TMZ, this was not the case for BCNU and CDDP; resistance to the latter drugs clearly involves processes other than the two DNA repair pathways analyzed in this study.

The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)-and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinasedead form of AKT1 into breast cancer cells abrogated TMZinduced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor D-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)-propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)-mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O 6 -guanine-methylating agents.The serine/threonine protein kinase AKT is a critical regulator of major cellular processes, including gene expression, glycogen metabolism, migration, proliferation, and survival (for review, see Bellacosa et al., 2005). Three isoforms of AKT, encoded by three different genes, are present in mammalian cells. Each isoform comprises an NH 2 -terminal pleckstrin homology domain, a catalytic protein kinase domain, and a short COOH-terminal region. All AKT isoforms contain two main regulatory phosphorylation sites, one within the kinase domain (Thr308/AKT1, Thr309/AKT2, Thr305/ AKT3), and one in the COOH-terminal region (Ser473/AKT1, Ser474/AKT2, Ser472/AKT3).This study was supported by the Italian Ministry of Health. Article, publication date, and citation information can be found at

The use of IFN-A in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-A also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-A, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8 + T cells recognizing modified and native MART-1 and gp100 peptides and MART-1 + gp100 + melanoma cells was observed. Moreover, vaccination induced an increase in CD8 + T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA + CCR7 À (terminally differentiated effectors) and

No abstract