Degradation of chromosomal DNA during apoptosis

Apoptosis is often accompanied by degradation of chromosomal DNA. CAD, caspase-activated DNase, was identified in 1998 as a DNase that is responsible for this process. In the last several years, mice deficient in the CAD system have been generated. Studies with these mice indicated that apoptotic DNA degradation occurs in two different systems. In one, the DNA fragmentation is carried out by CAD in the dying cells and in the other, by lysosomal DNase II after the dying cells are phagocytosed. Several other endonucleases have also been suggested as candidate effectors for the apoptotic degradation of chromosomal DNA. In this review, we will discuss the mechanism and role of DNA degradation during apoptosis.     

Thalictrum minus cell cultures and ABC-like transporter

Cultured Thalictrum minus cells produce a benzylisoquinoline alkaloid, berberine, in the presence of benzyladenine, and excrete it into the culture medium. T. minus cells excluded berberine, even if berberine was exogenously added to the medium, without benzyladenine treatment. Similarly, T. minus cells excluded a heterocyclic dye (neutral red) and calcein AM, which is used as a fluorescent probe to detect the drug efflux pump activity by ABC transporters. The addition of several inhibitors of P-glycoprotein, a representative ABC transporter, induced the accumulation in of both berberine and calcein AM ATP-dependent manner. The expression of P-glycoprotein-like ABC transporter genes was also demonstrated. The involvement of ABC transporter in the secretion of berberine in T. minus cells is discussed.     

Importance of renal mitochondria in the reduction of TEMPOL,a nitroxide radical

Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol.     

Apoptosis induction in HL-60 cells and inhibition of topoisomerase II by triterpene celastrol

Celastrol, which is a triterpene purified from Celastraceae plants, has anticancer and anti-inflammatory activities. In this study we investigated to clarify whether celastrol can induce apoptosis in a human leukemia HL-60 model system. Celastrol was found to induce apoptosis, and the rank order of the potency of celastrol and its derivatives to induce internucleosomal DNA fragmentation was found to be celastrol>cela-H>the other derivatives=vehicle control. Many anticancer agents are known to possess the ability to inhibit topoisomerase II, so the inhibitory activities of celastrol and its derivatives on topoisomerase II were also explored. The rank order of the inhibitory activity was found to be celastrol>etoposide>cela-H, indicating that the apoptosis-inducing activities of cela derivatives correspond to their inhibitory activities on topoisomerase II. These data suggested that celastrol may cause its effects such as anticancer activity by the mechanism of apoptosis along with topoisomerase II inhibition.     

Identification of the (183)RWTNNFREY(191) region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.     

Up-regulation of E-cadherin and β-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-α

Summary Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-α. E-cadherin expression was only up-regulated by butyrate and interferon-α (IFN-α) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, β-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-α. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that α-and γ-catenin expression was not changed, while only the expression of β-catenin increased. β-Catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type β-catenin are potentially modulated by butyrate and IFN-α, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.     

Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.     

New EIA technique for tyrosinase in human melanocytes and its application

The expression of tyrosinase in melanocytes relates to skin pigmentation or depigmentation. Although many types of drugs with whitening effects are well known, neither the definite effect nor the mechanism underlying the effect has been elucidated. In this study, we attempted to develop the rapid and simple EIA technique for tyrosinase protein, then this technique was applied to normal human cultured melanocytes. When primary antibody and tyrosinase were incubated in non-coated 96-well microtitre plates for 48 hours at 4 degrees C, then the solution in tyrosinase-coated plate was further incubated for another 1 hour at 37 degrees C. Thus the best results were obtained. The developed EIA system could detect authentic tyrosinase until 0.1-1.0 ng/mL. This EIA technique could also be applied to human cultured melanocytes. The melanocytes cultured with endothelin-1 induced tyrosinase like immune reactive protein. The protein induction with endothelin-1 was suppressed by BQ 123, ETa receptor antagonists. The simple EIA technique developed for tyrosinase may give a clue to determination of the onset mechanisms underlying pigmental diseases of the skin as well as the mechanisms underlying the effects of various whitening drugs.