Simultaneous induction of apoptosis and necroptosis by Tanshinone IIA in human hepatocellular carcinoma HepG2 cells.

Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plant Salvia miltiorrhiza BUNGE, has been reported to possess anticancer activity through induction of apoptosis in many cancer cells. Surprisingly, the present study finds that Tan IIA simultaneously causes apoptosis and necroptosis in human hepatocellular carcinoma HepG2 cells. We further find that apoptosis can be converted to necroptosis by pan-caspase inhibitor Z-VAD-fmk, and the two death modes can be blocked by necroptotic inhibitor necrostatin-1. The underlying mechanisms are revealed by analysis of the signaling molecules using western blotting. In control cells, FLICE inhibitory protein in short form (FLIPS) is expressed in relatively high levels and binds to caspase 8 in ripoptosome, which supposedly sustains cell survival. However, in Tan IIA-treated cells, FLIPS is down-regulated and may thus cause homodimer formation of cleaved caspase 8, cleavage of receptor-interacting serine/threonine-protein kinases 1, 3 (RIP1, RIP3), and mixed-lineage kinase domain-like (MLKL), in turn leads to cell apoptosis. In parallel, Tan IIA causes necroptosis by forming a suggested necrosomal complex composed of RIP1/RIP3. Regarding the inhibitors, z-VAD-fmk diminishes the cleaved caspase 8, RIP1, RIP3, and MLKL induced by Tan IIA, and reconstructs the ripoptosome complex, which marks cells moving from apoptosis to necroptosis. Nec-1 recovers the Tan IIA down-regulated FLIPS, consequently causes FLIPS to form heterodimer with caspase 8 and thus block apoptosis. Meanwhile, cleaved forms of RIP1 and RIP3 were observed preventing necroptosis. Intriguingly, the cytotoxicity of tumor necrosis factor-related apoptosis-inducing ligand to HepG2 cells is enhanced by Tan IIA in a pilot study, which may be attributed to low FLIPS levels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIPS regulation-mediated apoptosis/necroptosis, which has not been previously documented. Moreover, the involvement of the cleavage type of MLKL in executing necroptosis warrants further investigation.

The correlation between neurotoxicity, aggregative ability and secondary structure studied by sequence truncated Abeta peptides.

Aggregated beta-amyloid (Abeta) peptides are neurotoxic and cause neuronal death both in vitro and in vivo. Although the formation of a beta-sheet structure is usual required to form aggregates, the relationship between neurotoxicity and the Abeta sequence remains unclear. To explore the correlation between Abeta sequence, secondary structure, aggregative ability, and neurotoxicity, we utilized both full-length and fragment-truncated Abeta peptides. Using a combination of spectroscopic and cellular techniques, we demonstrated that neurotoxicity and aggregative ability are correlated while the relationship between these characteristics and secondary structure is not significant. The hydrophobic C-terminus, particularly the amino acids of 17-21, 25-35, and 41-42, is the main region responsible for neurotoxicity and aggregation. Deleting residues 17-21, 25-35 or 41-42 significantly reduced the toxicity. On the other hand, truncation of the peptides at either residues 22-24 or residues 36-40 had little effect on toxicity and aggregative ability. While the N-terminal residues 1-16 may not play a major role in neurotoxicity and aggregation, a lack of N-terminal fragment Abeta peptide, (e.g. Abeta17-35), does not display the neurotoxicity of either full-length or 17-21, 25-35 truncated Abeta peptides.

The SV40 small t-antigen prevents mammary gland differentiation and induces breast cancer formation in transgenic mice; truncated large T-antigen molecules harboring the intact p53 and pRb binding region do not have this effect.

We report here for the first time, that the SV40 small t-antigen inhibits mammary gland differentiation during mid-pregnancy and that about 10% of multiparous WAP-SVt transgenic animals develop breast tumors with latencies ranging from 10-17 months. Cyclin D1 is deregulated and over expressed in the small t-antigen positive mammary gland epithelial cells (ME-cells) and in the breast tumor cells. SV40 small t-antigen immortalized ME-cells (t-ME-cells) exhibit a strong intranuclear cyclin D1 staining, also in the absence of external growth factors and the cells continue to divide for several days without serum. In addition, the expression rate of cyclin E and p21(Waf1) but not of p53 is increased. Coimmunoprecipitation experiments revealed that p21(Waf1) is mainly associated with the cyclin D/CDK4 but not with the cyclin E/CDK2 complex. WAP-SVT transgenic animals exhibit an almost regular mammary gland development until late pregnancy but the majority of the ME-cells are eliminated by apoptosis during the early lactation period. Tumor formation is delayed and less efficient than in T/t-antigen positive animals. Sequestration of p53 and pRb by the N-terminal truncated T-antigen molecules (T1-antigen and T2-antigen) does not affect mammary gland differentiation and the transgenic animals (WAP-SVBst-Bam) do not develop breast tumors.

SV40 T/t-antigen induces premature mammary gland involution by apoptosis and selects for p53 missense mutation in mammary tumors.

We recently established transgenic animals (WAP-SV-T/t) carrying the early coding region of Simian Virus 40 (SV40) under the transcriptional control of the whey acidic milk protein promoter (WAP), which restricts the expression of the transgene to mammary gland epithelial cells (ME-cells). SV40 T/t-antigen synthesis causes premature mammary gland involution during late pregnancy by inducing apoptosis and leads to development of mammary tumors after the first lactation period in both p53 positive (WAP-SV-T/t) and p53 negative double transgenic animals (WAP-SV-T/t.p53-/-). The high apoptotic rate persists in all of the T/t-antigen positive breast tumor cells, as well as in established ME-tissue culture cell lines. ME-cells which spontaneously switch off the expression of the WAP-SV-T/t transgene do not undergo apoptosis. However, these cells again exhibit an extensive DNA fragmentation when SV40 T/t-antigen synthesis is reintroduced, which indicates that it is the expression of T/t antigen which is the critical factor for induction of apoptosis. In addition, we isolated several ME-cell lines from different breast tumors which have spontaneously lost the T/t-antigen yet remain maximally transformed. Strikingly, these cells contain a missense mutation of the p53 gene at codon 242 (p53(242)), which substitutes alanine for glycine. This mutation increases p53 stability and it reduces the transactivating function of p53, albeit without affecting the ability of the protein to interact with the DNA. This indicates that p53 missense mutations are selected for in breast tumors initially expressing T/t-antigen. Therefore, the p53(242) mutation is sufficient to maintain the transformed state after the ME-cells have switched off the WAP-SV-T/t transgene. Interestingly, the p53 minus state per se is not sufficient to induce ME-cell transformation since homozygous null mice for the p53 gene (p53-/-) fail to develop breast cancer.

The SV40 T-antigen induces premature apoptotic mammary gland involution during late pregnancy in transgenic mice.

Transgenic animals of the line 8 contain the WAP-SV-T transgene. Females of this line synthesise the SV40 T-antigen in mammary gland epithelial cells during pregnancy and the lactation period. All females are 'milk-less' and the offspring have to be nursed by foster mothers. The reason for this phenomenon is a premature apoptosis during late pregnancy. Nontheless a significant number of mammary epithelial cells escape apoptosis and all transgenic females devlop breast cancer after the first lactation period.

SV40 T-antigen induces breast cancer formation with a high efficiency in lactating and virgin WAP-SV-T transgenic animals but with a low efficiency in ovariectomized animals.

The whey acid protein (WAP) is a major mouse milk protein and its gene expression is induced by various lactotrophic hormones (eg, estrogen, progesterone). Transgenic animals harboring the early SV40 coding region (T/t-antigen) under the transcriptional control of the WAP promoter develop breast cancer after the first lactation period. The tumor cells synthesize the SV40 T-antigen with a high efficiency indicating that WAP-SV-T expression escapes down-regulation after the lactation period. However about 5-10% of the tumors became T-antigen negative during tumor progression and WAP-SV-T expression was only demonstrable by PCR analysis. Both T-antigen positive and negative tumor cells expressed the estrogen and progesterone receptor at a comparable rate, indicating that hormone receptor levels do not determine expression of the WAP-SV-T transgene. Furthermore, WAP and WAP-SV-T gene expression are not restricted to the pregnancy-lactation period. Virgin animals also express both genes with a low efficiency and about 70% of these animals also developed T-antigen positive breast tumors. The tumor rate however was strongly reduced in ovariectomized animals, indicating that the ovary hormones play a critical role in breast cancer formation.

Breast cancer formation in transgenic animals induced by the whey acidic protein SV40 T antigen (WAP-SV-T) hybrid gene.

After injection of the whey acidic protein (WAP)-SV-T hybrid gene into fertilized mouse eggs, eight independent transgenic mouse lines were obtained. Females from three lines developed mammary carcinomas with high frequency, coinciding mostly with lactation. In contrast to the endogenous WAP gene, expression of the hybrid gene continued after lactation. The tumor cells had a very invasive growth characteristic. Tumor regression in vivo was not observed. However, after transfer into tissue culture 25% of the cells ceased to express the hybrid gene and acquired the growth characteristic of normal cells. It was possible to retransform these cells by injection of wild-type SV40 DNA, but not after transfer of the hybrid WAP-SV-T gene. Inactivation of the endogenous WAP and of the WAP-SV-T transgene did not correlate with DNA methylation.

Sodium arsenite potentiates the clastogenicity and mutagenicity of DNA crosslinking agents.

To see if sodium arsenite enhances the clastogenicity and the mutagenicity of DNA crosslinking agents, Chinese hamster ovary (CHO) cells and human skin fibroblasts were exposed to cis-diamminedichloroplatinum (II) (cis-Pt(II)) or 8-methoxypsoralen (8-MOP) plus long-wave ultraviolet light (UVA) and then to sodium arsenite. The results indicate that the clastogenicity of cis-Pt(II) and 8-MOP plus UVA are enhanced by the post-treatment with sodium arsenite. Chromatid breaks and exchanges are predominantly increased in doubly treated cells. Furthermore, the mutagenicity of cis-Pt(II) at the hypoxanthine-guanine phosphoribosyl transferase locus is also potentiated by sodium arsenite in CHO cells.

Opposite staining effect of two silver-staining techniques on sister chromatids.

Opposite differential staining between sister chromatids was obtained by two silver-staining techniques on chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) and pretreated with Hoechst plus black light. Both silver-nitrate and silver-carbonate staining were affected by chemical extraction and enzyme digestion of chromosomal proteins. Prestaining of silver nitrate or silver carbonate also blocked the fluorescences of protein dyes. However, removal of chromosomal DNA affected the silver-carbonate but not the silver-nitrate staining; the fluorescences of DNA dyes were blocked by the prestaining of silver carbonate but not silver nitrate. Chromosomal protein labelling was released only slightly and its relative amount between BrdU bifilarly substituted and unifilarly substituted chromatids was unchanged during pretreatment of Hoechst plus black light. We speculate that chromosomal non-histones are the targets for silver-nitrate stain, and DNA-non-histone complexes for silver-carbonate stain.