In vivo silencing of Reptin blocks the progression of human hepatocellular carcinoma in xenografts and is associated with replicative senescence.

BACKGROUND & AIMS: We previously showed that Reptin is overexpressed in hepatocellular carcinoma (HCC), and that in vitro depletion of Reptin with siRNAs led to HCC cell growth arrest and apoptosis. Here, we asked whether in vivo targeting of Reptin in established tumours had a therapeutic effect. METHODS: We used lentiviral vectors to construct HuH7 and Hep3B cell lines with doxycycline (Dox)-dependent expression of Reptin (R2) or control shRNA (GL2). Cells were injected subcutaneously into immunodeficient mice, and Dox was given when tumours reached a volume of 250 mm(3). RESULTS: In vitro, the growth of GL2-Dox, GL2+Dox, and R2-Dox cells was undistinguishable whereas that of R2+Dox cells stopped 4 days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid beta-galactosidase and the presence of senescence-associated heterochromatin foci. In xenografted mice, R2+Dox tumour growth stagnated or even regressed with prolonged treatment in contrast with the GL2-Dox, GL2+Dox, and R2-Dox tumours that progressed steadily. The blockage of tumour progression was associated with the induction of senescence and reduced cell proliferation. CONCLUSIONS: In vivo Reptin depletion leads to tumour growth arrest. Reptin may prove a valuable target in HCC.

Two isoforms of human RNA polymerase III with specific functions in cell growth and transformation.

Transcription in eukaryotic nuclei is carried out by DNA-dependent RNA polymerases I, II, and III. Human RNA polymerase III (Pol III) transcribes small untranslated RNAs that include tRNAs, 5S RNA, U6 RNA, and some microRNAs. Increased Pol III transcription has been reported to accompany or cause cell transformation. Here we describe a Pol III subunit (RPC32beta) that led to the demonstration of two human Pol III isoforms (Pol IIIalpha and Pol IIIbeta). RPC32beta-containing Pol IIIbeta is ubiquitously expressed and essential for growth of human cells. RPC32alpha-containing Pol IIIalpha is dispensable for cell survival, with expression being restricted to undifferentiated ES cells and to tumor cells. In this regard, and most importantly, suppression of RPC32alpha expression impedes anchorage-independent growth of HeLa cells, whereas ectopic expression of RPC32alpha in IMR90 fibroblasts enhances cell transformation and dramatically changes the expression of several tumor-related mRNAs and that of a subset of Pol III RNAs. These results identify a human Pol III isoform and isoform-specific functions in the regulation of cell growth and transformation.

Adenosine triphosphatase pontin is overexpressed in hepatocellular carcinoma and coregulated with reptin through a new posttranslational mechanism.

UNLABELLED: Reptin and Pontin are related ATPases associated with stoichiometric amounts in several complexes involved in chromatin remodeling, transcriptional regulation, and telomerase activity. We found that Reptin was up-regulated in hepatocellular carcinoma (HCC) and that down-regulation of Reptin led to growth arrest. We show here that Pontin messenger RNA (mRNA) is also up-regulated in human HCC 3.9-fold as compared to nontumor liver (P = 0.0004). Pontin expression was a strong independent factor of poor prognosis in a multivariate analysis. As for Reptin, depletion of Pontin in HuH7 cells with small interfering RNAs (siRNAs) led to growth arrest. Remarkably, Pontin depletion led to down-regulation of Reptin as shown with western blot, and vice versa. Whereas siRNAs induced a decrease of their cognate mRNA targets, they did not affect the transcripts of the partner protein. Translation of Pontin or Reptin was not altered when the partner protein was silenced. However, pulse-chase experiments demonstrated that newly synthesized Pontin or Reptin stability was reduced in Reptin- or Pontin-depleted cells, respectively. This phenomenon was reversed upon inhibition of proteasome or ubiquitin-activating enzyme (E1). In addition, proteasome inhibition could partly restore Pontin steady-state levels in Reptin-depleted cells, as shown by western blot. This restoration was not observed when cells were also treated with cycloheximide, thus confirming that proteasomal degradation in this setting was restricted to newly synthesized Pontin. CONCLUSION: Reptin and Pontin protein levels are strictly controlled by a posttranslational mechanism involving proteasomal degradation of newly synthesized proteins. These data demonstrate a tight regulatory and reciprocal interaction between Reptin and Pontin, which may in turn lead to the maintenance of their 1:1 stoichiometry.

Pontin and reptin, two related ATPases with multiple roles in cancer.

Studies in model organisms or cultured human cells suggest potential implications in carcinogenesis for the AAA+ ATPases Pontin and Reptin. Both proteins are associated with several chromatin-remodeling complexes and have many functions including transcriptional regulation, DNA damage repair, and telomerase activity. They also interact with major oncogenic actors such as beta-catenin and c-myc and regulate their oncogenic function. We only now begin to get insight into the role of Pontin and Reptin in human cancers.

Protease-activated receptor 1 knockout reduces experimentally induced liver fibrosis.

Thrombin inhibition protects against liver fibrosis. However, it is not known whether the thrombin profibrogenic effect is due to effects on blood coagulation or to signaling via protease-activated receptors (PARs). We took advantage of the lack of blood coagulation defects in PAR-1-knockout mice. Acute carbon tetrachloride (CCl(4)) toxicity was similar in wild-type (WT), PAR-1(-/-), and PAR-1(+/-) mice as judged by aminotransferase levels, area of liver necrosis, and liver peroxidation measured by Fourier-transformed infrared spectroscopy. Fifteen mice/group received CCl(4) or its solvent for 6 wk (300 microl/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl(4) treatment in WT mice. PAR-1 deficiency protected against fibrosis, with 36% and 56% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively (P < 0.001). Similar results were obtained for area of activated fibrogenic cells (64% and 79% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively, P < 0.001). These findings were corroborated by measurements of type I collagen, matrix metalloproteinase-2, and PDGF-beta receptor mRNA levels. There was also a significant decrease in T lymphocyte infiltration in PAR-1-deficient mice. Altogether, these results suggest that thrombin profibrogenic effects are independent of effects on blood coagulation and are instead due to direct effects on fibrogenic cells and possibly on T lymphocytes.

Overexpression and role of the ATPase and putative DNA helicase RuvB-like 2 in human hepatocellular carcinoma.

UNLABELLED: Using a proteomic analysis of human hepatocellular carcinoma (HCC), we identified the overexpression in 4 tumors of RuvB-like 2 (RUVBL2), an ATPase and putative DNA helicase known to interact with beta-catenin and cellular v-myc myelocytomatosis viral oncogene homolog (c-myc). RUVBL2 expression was further analyzed in tumors with quantitative reverse-transcription polymerase chain reaction analysis and immunohistochemistry; in addition, RUVBL2 expression in a HuH7 cell line was silenced by small interfering RNA or increased with a lentiviral vector. RUVBL2 messenger RNA overexpression was confirmed in 72 of 96 HCC cases, and it was associated with poorly differentiated tumors (P = 0.02) and a poor prognosis (P = 0.02) but not with beta-catenin mutations or c-myc levels. Although RUVBL2 was strictly nuclear in normal hepatocytes, tumoral hepatocytes exhibited additional cytoplasmic staining. There was no mutation in the coding sequence of RUVBL2 in 10 sequenced cases. Silencing RUVBL2 in HuH7 HCC cells reduced cell growth (P < 0.001) and increased apoptosis, as shown by DNA fragmentation (P < 0.001) and caspase 3 activity (P < 0.005). This was associated with an increased expression of several proapoptotic genes and with an increased conformational activation of Bak-1 and Bax. On the other hand, HuH7 cells with an overexpression of RUVBL2 grew better in soft agar (P < 0.03), had increased resistance to C2 ceramide-induced apoptosis (P < 0.001), and gave rise to significantly larger tumors when injected into immunodeficient Rag2/gammac mice (P = 0.016). CONCLUSION: RUVBL2 is overexpressed in a large majority of HCCs. RUVBL2 overexpression enhances tumorigenicity, and RUVBL2 is required for tumor cell viability. These results argue for a major role of RUVBL2 in liver carcinogenesis.

Dissecting regulatory networks by means of two-dimensional gel electrophoresis: application to the study of the diauxic shift in the yeast Saccharomyces cerevisiae.

Using a proteomic approach based on the two-dimensional (2-D) gel analysis of synthesized proteins, we investigated the involvement of the Snf1 kinase pathway in the regulation of gene expression during the diauxic shift in Saccharomyces cerevisiae. For this purpose, we used a mutant strain deleted for SNF4, the gene coding for the activator subunit of Snf1p. The levels of synthesis of 82 spots were found to be affected by the absence of Snf4p at the diauxic shift. Half of the proteins which exhibit a reduced synthesis in the mutant strain are proteins whose genes are controlled by the transcriptional activator Cat8p, a target of Snf1p. Proteins with an increased level of synthesis in the mutant strain were also observed. Among them are glycolytic enzymes whose synthesis is strongly reduced when wild-type cells enter the diauxic shift. This observation suggests that Snf1p exerts a negative control on the expression of glycolytic genes during the diauxic transition. The results obtained in this study were compiled with those previously obtained by similar proteomic approach with other regulatory factors involved in the diauxic shift. This compilation illustrates how 2-D gel electrophoresis can be used to elucidate the network of regulators participating to complex biological process.

The Snf1 protein kinase controls the induction of genes of the iron uptake pathway at the diauxic shift in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the transition between the fermentative and the oxidative metabolism, called the diauxic shift, is associated with major changes in gene expression. In this study, we characterized a novel family of five genes whose expression is induced during the diauxic shift. These genes, FET3, FTR1, TIS11, SIT1, and FIT2, are involved in the iron uptake pathway. We showed that their induction at the diauxic shift is positively controlled by the Snf1/Snf4 kinase pathway. The transcriptional factor Aft1p, which is known to control their induction in response to iron limitation, is also required for their induction during the diauxic shift. The increase of the extracellular iron concentration does not affect this induction, indicating that glucose exhaustion by itself would be the signal. The possibility that the Snf1/Snf4 pathway was also involved in the induction of the same set of genes in response to iron starvation was considered. We demonstrate here that this is not the case. Thus, the two signals, glucose exhaustion and iron starvation, use two independent pathways to activate the same set of genes through the Aft1p transcriptional factor.