Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): evaluation using an endogenous HCC model

Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality and poor prognosis. Oncogenic transcription factor Late SV40 Factor (LSF) plays an important role in promoting HCC. A small molecule inhibitor of LSF, Factor Quinolinone Inhibitor 1 (FQI1), significantly inhibited human HCC xenografts in nude mice without harming normal cells. Here we evaluated the efficacy of FQI1 and another inhibitor, FQI2, in inhibiting endogenous hepatocarcinogenesis. HCC was induced in a transgenic mouse with hepatocyte-specific overexpression of c-myc (Alb/c-myc) by injecting N-nitrosodiethylamine (DEN) followed by FQI1 or FQI2 treatment after tumor development. LSF inhibitors markedly decreased tumor burden in Alb/c-myc mice with a corresponding decrease in proliferation and angiogenesis. Interestingly, in vitro treatment of human HCC cells with LSF inhibitors resulted in mitotic arrest with an accompanying increase in CyclinB1. Inhibition of CyclinB1 induction by Cycloheximide or CDK1 activity by Roscovitine significantly prevented FQI-induced mitotic arrest. A significant induction of apoptosis was also observed upon treatment with FQI. These effects of LSF inhibition, mitotic arrest and induction of apoptosis by FQI1s provide multiple avenues by which these inhibitors eliminate HCC cells. LSF inhibitors might be highly potent and effective therapeutics for HCC either alone or in combination with currently existing therapies.The present study was supported in part by grants from The James S. McDonnell Foundation, National Cancer Institute Grant R01 CA138540-01A1 (DS), National Institutes of Health Grant R01 CA134721 (PBF), the Samuel Waxman Cancer Research Foundation (SWCRF) (DS and PBF), National Institutes of Health Grants R01 GM078240 and P50 GM67041 (SES), the Johnson and Johnson Clinical Innovation Award (UH), and the Boston University Ignition Award (UH). JLSW was supported by Alnylam Pharmaceuticals, Inc. DS is the Harrison Endowed Scholar in Cancer Research and Blick scholar. PBF holds the Thelma Newmeyer Corman Chair in Cancer Research. The authors acknowledge Dr. Lauren E. Brown (Dept. Chemistry, Boston University) for the synthesis of FQI1 and FQI2, and Lucy Flynn (Dept. Biology, Boston University) for initially identifying G2/M effects caused by FQI1. (James S. McDonnell Foundation; R01 CA138540-01A1 - National Cancer Institute; R01 CA134721 - National Institutes of Health; R01 GM078240 - National Institutes of Health; P50 GM67041 - National Institutes of Health; Samuel Waxman Cancer Research Foundation (SWCRF); Johnson and Johnson Clinical Innovation Award; Boston University Ignition Award; Alnylam Pharmaceuticals, Inc.)Published versio

CD4-Independent Human Immunodeficiency Virus Infection Involves Participation of Endocytosis and Cathepsin B

During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1) strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1

Leaf litter traits of invasive alien species slow down decomposition compared to Spanish natives: a broad phylogenetic comparison.

Leaf traits related to the performance of invasive alien species can influence nutrient cycling through litter decomposition. However, there is no consensus yet about whether there are consistent differences in functional leaf traits between invasive and native species that also manifest themselves through their "after life" effects on litter decomposition. When addressing this question it is important to avoid confounding effects of other plant traits related to early phylogenetic divergences and to understand the mechanism underlying the observed results to predict which invasive species will exert larger effects on nutrient cycling. We compared initial leaf litter traits, and their effect on decomposability as tested in standardized incubations, in 19 invasive-native pairs of co-familial species from Spain. They included 12 woody and seven herbaceous alien species representative of the Spanish invasive flora. The predictive power of leaf litter decomposition rates followed the order: growth form > family > status (invasive vs. native) > leaf type. Within species pairs litter decomposition tended to be slower and more dependent on N and P in invaders than in natives. This difference was likely driven by the higher lignin content of invader leaves. Although our study has the limitation of not representing the natural conditions from each invaded community, it suggests a potential slowing down of the nutrient cycle at ecosystem scale upon invasion. © Springer-Verlag 2009

Inhibition of viral and cellular promoters by human wild-type p53.

Mutation of the p53 tumor suppressor gene is a recurring event in a variety of human cancers. Wild-type p53 may regulate cell proliferation and has recently been shown to repress transcription from several cellular promoters. We studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen promoter and on several viral promoters including the simian virus 40 early promoter-enhancer, the herpes simplex virus type 1 thymidine kinase and UL9 promoters, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus, human immunodeficiency virus type 1, and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and plasmids containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. Expression of wild-type p53 correlated with a consistent and significant (6- to 76-fold) reduction of reporter enzyme activity. A mutation at amino acid 143 of p53 releases this inhibition significantly with all the promoters studied. Expression of a p53 mutated at any one of the five amino acid positions 143, 175, 248, 273, and 281 also correlated with a much smaller (one- to sixfold) reduction of reporter enzyme activity from the herpes simplex virus type 1 thymidine kinase promoter. These mutant forms of p53 are found in various cancer cells. Thus, failure of tumor suppression correlates with loss of the promoter inhibitory effect of p53

Activation of the human immunodeficiency virus type 1 long terminal repeat by transforming mutants of human p53.

We have studied the effects of human wild-type and mutant p53s on the long terminal repeat (LTR) promoter of human immunodeficiency virus type 1 (HIV). HeLa cells were cotransfected with a wild-type or mutant p53 expression plasmid and a plasmid containing a chloramphenicol acetyltransferase reporter gene under HIV LTR promoter control. As expected, expression of wild-type p53 inhibited promoter function. Expression of a p53 mutated at any one of the four amino acid positions 175, 248, 273, and 281 correlated with a significant increase of the HIV promoter activity. The HIV LTR was also significantly activated in Saos-2 cells that do not express endogenous p53. This finding suggests a gain-of-transactivation function by mutation of the p53 gene. Cotransfection of wild-type and mutant p53-281G expression plasmids indicated that either the wild type or the mutant was dominant in inhibiting or enhancing promoter activity, respectively, when transfected in excess of the other. Transfection experiments showed transactivation even when the Sp1, NF-kappa B, and TATA sites in the LTR were individually mutated. Synthetic minimal promoter constructs containing two Sp1 sites or two NF-kappa B sites or an ATF site are also significantly activated by the mutant p53-281G. Thus, the mutant protein may activate transcription through interaction with either a general transcription factor or a common factor that bridges the basal transcription machinery and the transcription factors Sp1, NF-kappa B, and ATF