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We have identified a novel interleukin (IL)-7-responsive T cell population [forkhead box P3 (FoxP3(+) ) CD4(+) CD25(+) CD127(+) ] that is comparably functionally suppressive to conventional FoxP3(+) CD4(+) CD25(+) regulatory T cells (T(regs) ). Although IL-2 is the most critical cytokine for thymic development of FoxP3(+) T(regs) , in the periphery other cytokines can be compensatory. CD25(+) CD127(+) T cells treated with IL-7 phenotypically 'matured' into the known 'classical' FoxP3(+) CD4(+) CD25(high) CD127(-) FoxP3(+) T(regs) . In freshly isolated splenocytes, the highest level of FoxP3 expression was found in CD127(+) CD25(+) T cells when compared with CD127(-) CD25(+) or CD127(+) CD25(-) cells. IL-7 treatment of CD4(+) CD25(+) T cells induced an increase in the accumulation of FoxP3 in the nucleus in vitro. IL-7-mediated CD25 cell surface up-regulation was accompanied by a concurrent down-regulation of CD127 in vitro. IL-7 treatment of the CD127(+) CD25(+) FoxP3(+) cells also resulted in up-regulation of cytotoxic T lymphocyte antigen 4 without any changes in CD45RA at the cell surface. Collectively, these data support emerging evidence that FoxP3(+) T cells expressing CD127 are comparably functionally suppressive to CD25(+) CD127(-) FoxP3(+) T cells. This IL-7-sensitive regulation of FoxP3(+) T(reg) phenotype could underlie one peripheral non-IL-2-dependent compensatory mechanism of T(reg) survival and functional activity, particularly for adaptive T(regs) in the control of autoimmunity or suppression of activated effector T cells.

Modulating PI3K at different stages of dendritic cells (DC) generation could be a novel means to balance the generation of immunosuppressive versus immunostimulatory DC. We show that PI3K inhibition during mouse DC generation in vitro results in cells that are potently immunosuppressive and characteristic of CD8alpha- CD11c+ CD11b+ DC. These DC exhibited low surface class I and class II MHC, CD40, and CD86 and did not produce TNF-alpha. In allogeneic MLR, these DC were suppressive. Although in these mixed cultures, there was no increase in the frequency of CD4+ CD25+ Foxp3+ cells, the Foxp3 content on a per cell basis was significantly increased. Sustained TLR9 signaling in the presence of PI3K inhibition during DC generation overrode the cells' suppressive phenotype.

Paclitaxel (PTX) and beta-lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 microM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p-Akt levels and in ps166-MDM2, the phosphorylated-MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt-MDM2-p53 complex and increased nuclear p53 levels. Our results suggest that phospho-Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho-Akt and inhibitor of apoptosis proteins.

This paper shows that the histone deacetylase inhibitor SAHA sensitised at sub-toxic doses human hepatocellular carcinoma cells (HepG2, Hep3B and SK-Hep1) to TRAIL-induced apoptosis, while it was ineffective in primary human hepatocytes (PHHs). In particular in HCC cells SAHA increased the expression of death receptor 5 (DR5) and caused a decrement of c-Flip. These two modifications provoked in the presence of TRAIL the rapid production of TRAIL-DISC and the activation of caspase-8. Consequently SAHA/TRAIL combination induced many apoptotic events, such as a cleavage of Bid into tBid, dissipation of mitochondrial membrane potential, activation of caspase-3 with the consequent cleavage of both NF-kB and Akt. The decrease in NF-kB level seemed to be responsible for the reduction in the content of IAP family antiapoptotic proteins while the decrease in Akt level caused a reduction in phospho-Bad. These events led to the activation of caspase-9, which contributed to the strong apoptotic activity of TRAIL. Sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by SAHA may suggest new strategies for the treatment of liver tumours.

Partenolide, a natural compound used in traditional medicine for its anti-inflammatory activity, has recently shown anti-tumor and apoptotic effects. Our studies demonstrated that HepG2; Hep3B and SK-Hep1 hepatocarcinoma cells, which are resistant to human recombinant TRAIL, are potently sensitized to TRAIL-induced apoptosis by low doses of parthenolide resulting in a marked synergist effect. To clarify the mechanism that accounts for this interaction, we demonstrated that parthenolide/TRAIL combination markedly increased DR4 and DR5. These effects might be correlated with STAT proteins modifications. In fact parthenolide and parthenolide/TRAIL combination decreased STAT3 and STAT5 and their phosphorylated forms. These lowering effects could be responsible for increased expression of death receptors, as suggested by the observation that down-regulation of STAT proteins by siRNA, stimulated the expression of both DR4 and DR5. Although the anti-tumor activity of parthenolide was identified recently, this research area appears promising as it is likely that parthenolide/TRAIL combination may contribute to the rational design of novel targeted therapies.

This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.

Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. This paper shows that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) stimulated at 5-10 microM apoptosis in human hepatoma HepG2 and Huh6 cells, but was ineffective in primary human hepatocytes (PHH). In HepG2 cells SAHA induced the extrinsic apoptotic pathway, increasing the expression of both FasL and FasL receptor and causing the activation of caspase-8. Moreover, SAHA enhanced the level of Bim proteins, stimulated alternative splicing of the Bcl-X transcript with the expression of the proapoptotic Bcl-Xs isoform, induced degradation of Bid into the apoptotic factor t-Bid and dephosphorylation and inactivation of the anti-apoptotic factor Akt. Consequently, SAHA caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, activation of caspase-3 and degradation of PARP. Interestingly, a combination of suboptimal doses of SAHA (1 microM) and bortezomib (5-10 nM), a potent inhibitor of 26S proteasome, synergistically induced apoptosis in both HepG2 and Huh6 cells, but was ineffective in PHH. Combined treatment increased with synergistic effects the expression levels of c-Jun, phospho-c-Jun and FasL and the production of Bcl-Xs. These effects were accompanied by activation of Bid, caspase-8 and 3. In conclusion, SAHA stimulated apoptosis in hepatoma cells and exerted a synergistic apoptotic effect when combined with bortezomib. In contrast, these treatments were quite ineffective in inducing apoptosis in PHH. Thus, our results suggest the potential application of the SAHA/bortezomib combination in clinical trials for liver cancer.

This review, in addition to updating the growing list of type 1 diabetes- relevant gene therapies, offers an outline of short-term objectives that can readily be met to move, at least, adenoviral and adeno-associated viral-based protocols into the clinic, first as a means of facilitating islet allografts as well as platforms with which to introduce immunoregulatory transgenes. A wide array of genes have been tested to restore insulin production, to drive the differentiation of insulin-producing progenitors, and to confer immunosuppression in an antigen- and tissue-restricted manner.

Butyrate can promote programmed cell death in a number of tumour cells in vitro. This paper provides evidence that butyrate induces apoptosis in human hepatoma HuH-6 and HepG2 cells but is ineffective in Chang liver cells, an immortalised non-tumour cell line. In both HuH-6 and HepG2 cells, apoptosis appeared after a lag period of approximately 16 h and increased rapidly during the second day of treatment. In particular, the effect was stronger in HuH-6 cells, which were, therefore, chosen for ascertaining the mechanism of butyrate action. In HuH-6 cells, beta-catenin seemed to exert an important protective role against apoptosis, since pretreatment with beta-catenin antisense ODN reduced the content of beta-catenin and anticipated the onset of apoptosis at 8 h of exposure to butyrate. Moreover, in HuH-6 cells, butyrate induced loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspase 9 and caspase 3, and degradation of poly(ADP-ribose) polymerase. In addition, during the second day of treatment, beta-catenin, pRb, and cyclins D and E were diminished and the phosphorylated form of pRb disappeared. Also, the content of the anti-apoptotic factor Bcl-XL fell markedly during this period, while that of the pro-apoptotic factor Bcl-Xs increased. These effects were accompanied by an increase in both Bcl-XL and Bcl-Xs mRNA transcripts, as ascertained by reverse transcriptase-polymerase chain reaction. Our results suggest that caspases have a crucial role in butyrate-induced apoptosis. This conclusion is supported by the observation that the inhibitors of caspases, benzyloxy carbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxy carbonyl-Asp-Glu-Val-Asp-fluoromethylketone, prevented apoptosis and the decrease in Bcl-XL, pRb, cyclins and beta-catenin. These effects were most probably responsible for the increased sensitivity of the cells to butyrate-induced apoptosis, which was observed on the second day of treatment.

A potent inhibitor of serine/threonine kinases, staurosporine exerts antiproliferative and apoptotic effects in many cancer cells, although the exact mechanism of its action is still unclear. This study examines the effects of staurosporine on Chang liver cells, an immortalized non-tumor cell line, in comparison with those caused in HuH-6 and HepG2 cells, two human hepatoma cell lines. Our results provide evidence that staurosporine promotes apoptosis in Chang liver cells as observed by flow cytometric analysis and acridine orange/ethidium bromide staining. The effect appeared already after 8 h of treatment and increased with treatment time and dose. After 48 h of exposure to 200 nM staurosporine clear apoptotic signs were observed in about 50% of the cells. Western blotting analysis showed that in Chang liver cells staurosporine induced a marked decrease in the levels of the antiapoptotic factors Bcl-2 (-75%) and Bcl-XL (-50%). Staurosporine also caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3. The involvement of caspases in staurosporine-induced cell death was also suggested by the observation that the addition of z-VAD-fmk, a general inhibitor of caspases, suppressed apoptosis. In HuH-6 and HepG2 cells treatment with staurosporine induced the arrest of cells in G2/M phase of cell cycle. This effect was not modified by z-VAD-fmk and was not accompanied by the appearance of biochemical signs of apoptosis. We conclude that staurosporine induced apoptosis in Chang liver cells by a mitochondria-caspase-dependent pathway which was closely correlated with a decrease in Bcl-2 and Bcl-XL levels, while in HuH-6 and HepG2 hepatoma cells the drug caused only an antiproliferative effect.

This study demonstrates that in human osteosarcoma cells treatment with 3-aminobenzamide (3-AB), a potent inhibitor of poly(ADP-ribose) polymerase (PARP), induces morphological and biochemical features of differentiation, the duration of which depends on whether or not the normal RB gene is expressed. In Saos-2 cells expressing a non-functional Rb protein, 3-AB treatment induced the formation of transient, short dendritic-like protrusions. In RB-transfected-Saos-2 cells (a clone previously generated in our laboratory that shows stable expression of wild-type Rb protein), 3-AB induced marked and prolonged changes with the formation of long dendritic-like protrusions and the appearance of stellate (osteocyte-like) cells. In MG-63 cells producing a wild-type Rb protein, 3-AB treatment had more marked effects, with a larger number of cells assuming the stellate appearance of osteocytes, which were connected to each other via junctions resembling small channels. Regardless of cell type, at some point after 3-AB treatment the differentiative attempt failed and the cells died. Death was apoptotic, as demonstrated by chromatin condensation and fragmentation, specific cleavage of PARP and Lamin-B, processing of caspase-3 and the appearance of Bax immunoreactive species. Enzymatic assay and RT-PCR of alkaline phosphatase (ALP) - an enzyme whose levels markedly decrease when osteoblasts undergo terminal differentiation into osteocytes - showed that 3-AB treatment markedly lowered ALP expression. Simultaneously, 3-AB treatment markedly increased the expression of CD44, a transmembrane multifunctional adhesion molecule and sensitive marker of osteocytic differentiation. This study hypothesizes a cross-talk between pRb and PARP and suggests that PARP may be a useful target for anticancer drugs.

Induction of apoptosis in human osteosarcoma Saos-2 cells by the proteasome inhibitor MG132 and the protective effect of pRb

This report is focused on the apoptotic effect induced by MG132, an inhibitor of 26S proteasome, in human hepatoma HepG2 cells. The results were compared with those obtained with non-transformed human Chang liver cells. MG132 reduced the viability of HepG2 cells in a time- and dose-dependent manner. The effect was in tight connection with the induction of apoptosis, as indicated by fluorescence microscopy and cytometric analysis, and was accompanied by a remarkable increase in the production of H2O2 and a reduction in mitochondrial transmembrane potential (Deltapsim). In addition cell death was prevented by antioxidants such as GSH, N-acetylcysteine or catalase. Western blot analysis showed that HepG2 cells contain a very low level of Bcl-2 and a much higher level of Bcl-XL, another antiapoptotic factor of the same family. When the cells were exposed to MG132 the level of Bcl-XL diminished, while a new band, corresponding to the expression of the proapoptotic protein Bcl-XS was detected. MG132 also caused the release of cytochrome c from mitochondria and the activation of caspase-3 with the consequent degradation of poly-ADP ribose polymerase (PARP). The observation that the broad spectrum caspase inhibitor z-VAD markedly reduced the apoptotic effect of the drug clearly demonstrated that caspases play an important role in MG132-induced apoptosis. MG132 exerted a modest effect on the viability of Chang liver cells which primarily depended on the G2/M arrest of cell cycle while only a small percentage of apoptotic cells was found. The remarkable differences in the effects induced by MG132 in Chang liver cells and HepG2 cells made us hypothesise the potential use of proteasome inhibitors in hepatocarcinoma therapy.

Our results demonstrate that sodium phenylbutyrate, a compound with a low degree of toxicity, exerted a cytotoxic effect on human retinoblastoma Y79 cells in a time- and dose-dependent manner. Treatment of Y79 cells for 72 h with phenylbutyrate reduced cell viability by 63% at 2 mM and 90% at 4 mM. Cell death caused by phenylbutyrate exhibited the typical features of apoptosis, as shown by light and fluorescent microscopy. Western blot analysis demonstrated that exposure of Y79 cells to phenylbutyrate decreased the level of the antiapoptotic factor Bcl-2 and induced the activation of caspase-3, a key enzyme in the execution phase of apoptosis. Moreover, treatment with phenylbutyrate markedly increased the level of acetylated histone-H3. Combined treatment with phenylbutyrate and topotecan, a topoisomerase I-inhibitor, resulted in a clear synergistic effect. We suggest that the effects exerted by phenylbutyrate on Y79 cells essentially depend on modifications of gene expression consequent to histone hyperacetylation.

Loss of RB1 gene is considered either a causal or an accelerating event in retinoblastoma. A variety of mechanisms inactivates RB1 gene, including intragenic mutations, loss of expression by methylation and chromosomal deletions, with effects which are species-and cell type-specific. RB1 deletion can even lead to aneuploidy thus greatly increasing cancer risk. The RB1gene is part of a larger gene family that includes RBL1 and RBL2, each of the three encoding structurally related proteins indicated as pRb, p107, and p130, respectively. The great interest in these genes and proteins springs from their ability to slow down neoplastic growth. pRb can associate with various proteins by which it can regulate a great number of cellular activities. In particular, its association with the E2F transcription factor family allows the control of the main pRb functions, while the loss of these interactions greatly enhances cancer development. As RB1 gene, also pRb can be functionally inactivated through disparate mechanisms which are often tissue specific and dependent on the scenario of the involved tumor suppressors and oncogenes. The critical role of the context is complicated by the different functions played by the RB proteins and the E2F family members. In this review, we want to emphasize the importance of the mechanisms of RB1/pRb inactivation in inducing cancer cell development. The review is divided in three chapters describing in succession the mechanisms of RB1 inactivation in cancer cells, the alterations of pRb pathway in tumorigenesis and the RB protein and E2F family in cancer.