Induction of apoptosis and CD10/neutral endopeptidase expression by jaspamide in HL-60 line cells

Jaspamide (jasplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo, its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells. Received 22 April 2002; received after revision 8 June 2002; accepted 10 June 2002     

Emerging connections between DNA methylation and histone acetylation

Modifications of both DNA and chromatin can affect gene expression and lead to gene silencing. Evidence of links between DNA methylation and histone hypoacetylation is accumulating. Several proteins that specifically bind to methylated DNA are associated with complexes that include histone deacetylases (HDACs). In addition, DNA methyltransferases of mammals appear to interact with HDACs. Experiments with animal cells have shown that HDACs are responsible for part of the repressive effect of DNA methylation. Evidence was found in Neurospora that protein acetylation can in some cases affect DNA methylation. The available data suggest that the roles of DNA methylation and histone hypoacetylation, and their relationship with each other, can vary, even within an organism. Some open questions in this emerging field that should be answered in the near future are discussed.     

Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis

Cancer cells are typically characterized by apoptosis deficiency. In order to investigate a possible role for the anti-apoptotic livin gene in renal cell cancer (RCC), we analyzed its expression in tumor tissue samples and in RCC-derived cell lines. In addition, we studied the contribution of livin to the apoptotic resistance of RCC cells by RNA interference (RNAi). Livin gene expression was detected in a significant portion of RCC tumor tissue specimens (13/14, 92.9%) and tumor-derived cell lines (12/15, 80.0%). Moreover, targeted inhibition of livin by RNAi markedly sensitized RCC cells towards proapoptotic stimuli, such as UV irradiation or the chemotherapeutic drugs etoposide, 5-fluorouracil, and vinblastine. These effects were specific for livin expressing tumor cells. We conclude that livin can contribute significantly to the apoptosis resistance of RCC cells. Targeted inhibition of livin could represent a novel therapeutic strategy to increase the sensitivity of renal cancers towards pro-apoptotic agents. Received 30 November 2006; received after revision 22 February 2007; accepted 20 March 2007     

Rapid reversion of aging phenotypes by nicotinamide through possible modulation of histone acetylation

Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated β-galactosidase activity. Although NAA seems to enhance the replicative potential of the cells, it has little effect on their growth rate and life span, suggesting that NAA action is rather separated from the cellular replicative system. The effects are unique to NAA: none of the NAA-related compounds examined (an NAD precursor/niacin, NAD analogs, and poly(ADP-ribose) polymerase inhibitors) exerted similar effects. Thus, NAD-related metabolism and poly(ADP-ribosyl)ation are unlikely related to the NAA action. On the other hand, histone acetyltransferase (HAT) activity was elevated in NAA-exposed cells, while in aged cells, HAT activity and histone H4 acetylation were lowered. Taken together, the results suggest that NAA may cause rejuvenation by restoring, at least in part, altered gene expression in aged cells through its activation of HAT. Received 27 August 2001; received after revision 15 October 2001; accepted 15 October 2001     

Extensive characterization of sphere models established from colorectal cancer cell lines

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of “stemness”. These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.     

Proteasome inhibition overcomes the resistance of renal cell carcinoma cells against the PPARγ ligand troglitazone

In order to analyze the effects of peroxisome proliferator-activated receptor-γ (PPARγ) activation on renal cell carcinomas we utilized several cell lines that were treated with the high affinity PPARγ agonist, troglitazone. Incubation of RCC cells with troglitazone resulted in reduced secretion of growth factors that was due to the inhibition of MAP kinase signaling and reduced nuclear localized expression of relB and HIF1alpha. Interestingly, the cell lines used showed a different sensitivity towards apoptosis induction that did not correlate with the inhibition of growth factors or expression of pro- and antiapoptotic molecules. To overcome this resistance the cells were treated with a combination of troglitazone and the proteasome inhibitor, bortezomib. The combination of both compounds induced apoptosis even in cells resistant to both agents alone, due to increased induction of ER-stress and caspase-3 mediated cell death. Received 03 September 2009; received after revision 02 February 2009; accepted 10 February 2009     

New frontiers of primary antibody deficiencies

Primary antibody deficiencies (PAD) form the largest group of inherited disorders of the immune system. They are characterized by a marked reduction or absence of serum immunoglobulins (Ig) due to disturbed B cell differentiation and by a poor response to vaccination. PAD can be divided into agammaglobulinemia, Ig class switch recombination deficiencies, and idiopathic hypogammaglobulinemia. Over the past 20 years, defects have been identified in 18 different genes, but in many PAD patients the underlying gene defects have not been found. Diagnosis of known PAD and discovery of new PAD is important for good patient care. In this review, we present the effects of genetic defects in the context of normal B cell differentiation, and we discuss how new technical developments can support understanding and discovering new genetic defects in PAD.     

MDR1, cholesterol certification and cell growth: a comparative study in normal and multidrug-resistant KB cell lines

The product of the MDR1 gene (P-gp) has been implicated in the transport of cholesterol from plasma membrane to endoplasmic reticulum for esterification. In previous studies on leukemia cell lines, we suggested that cholesterol esterification may regulate the rate of cell growth and that the MDR1 gene might be involved in this process by modulating intracellular cholesterol esters levels. To further investigate this matter, the rate of cell growth, cholesterol metabolism, expression of the MDR1 gene, and P-gp activity were compared in KB cell lines displaying differences in expression and function of P-gp (drug-sensitive phenotype versus MDR phenotype). The rate of cell growth correlated with cholesterol esterification in all KB cell lines, whereas the over-expression of MDR1 observed in the MDR cell lines was not always associated with an increased capacity of cells to esterify cholesterol. Two known inhibitors of P-gp activity, progesterone and verapamil, strongly inhibited both cholesterol esterification and cell proliferation in all KB cell lines, but they affected intracellular accumulation of labeled vinblastine only in MDR cell lines. These results further support a role for cholesterol esters in the regulation of cell growth and suggest that the P-gp expressed in MDR KB cells is not involved in the general process leading to cholesterol esterification. Received 14 February 2000; received after revision 10 April 2000; accepted 8 May 2000     

JNK1-dependent antimitotic activity of thiazolidin compounds in human non-small-cell lung and colon cancer cells

We recently identified two thiazolidin compounds, 5-[(4-methylphenyl)methylene]-2-(phenylamino)-4(5H)-thiazolone (MMPT) and 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin (DBPT), that inhibit the growth of human non-small-cell lung and colon cancer cells independent of P-glycoprotein and p53 status. Here we further investigated the mechanism by which these thiazolidin compounds mediate their anticancer effects. Treatment of cancer cells with MMPT and DBPT led to a time-dependent accumulation of cells arrested in the G2/M phase with modulation of the expression of proteins such as cyclin B1, cdc25C, and phosphorylated histone H3. Moreover, treatment with MMPT and DBPT increased M-phase arrest with abnormal spindle formation. DBPT-mediated G2/M phase arrest and phosphorylation of cdc25C and histone H3 were abrogated when JNK activation was blocked either with SP600125, a specific JNK inhibitor, or a dominant-negative JNK1 gene. Moreover, DBPT-mediated microtubule disruption was also blocked by SP600125 treatment. Our results demonstrate that thiazolidin compounds can effectively induce G2/M arrest in cancer cells and that this G2/M arrest requires JNK activation.     

Histone variants: emerging players in cancer biology

Histone variants are key players in shaping chromatin structure, and, thus, in regulating fundamental cellular processes such as chromosome segregation and gene expression. Emerging evidence points towards a role for histone variants in contributing to tumor progression, and, recently, the first cancer-associated mutation in a histone variant-encoding gene was reported. In addition, genetic alterations of the histone chaperones that specifically regulate chromatin incorporation of histone variants are rapidly being uncovered in numerous cancers. Collectively, these findings implicate histone variants as potential drivers of cancer initiation and/or progression, and, therefore, targeting histone deposition or the chromatin remodeling machinery may be of therapeutic value. Here, we review the mammalian histone variants of the H2A and H3 families in their respective cellular functions, and their involvement in tumor biology.     

Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils

Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6-24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48-120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48-120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.