Piotr Widlak

Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G

Toward the end of the 20th and beginning of the 21st centuries, clever in vitro biochemical complementation experiments and genetic screens from the laboratories of Xiaodong Wang, Shigekazu Nagata, and Ding Xue led to the discovery of two major apoptotic nucleases, termed DNA fragmentation factor (DFF) or caspase‐activated DNase (CAD) and endonuclease G (Endo G). Both endonucleases attack chromatin to yield 3′‐hydroxyl groups and 5′‐phosphate residues, first at the level of 50–300 kb cleavage products and next at the level of internucleosomal DNA fragmentation, but these nucleases possess completely different cellular locations in normal cells and are regulated in vastly different ways. In non‐apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45) [also called inhibitor of CAD (ICAD‐L)] and a 40 kD latent nuclease subunit (DFF40/CAD). Apoptotic activation of caspase‐3 or ‐7 results in the cleavage of DFF45/ICAD and release of active DFF40/CAD nuclease. DFF40s nuclease activity is further activated by specific chromosomal proteins, such as histone H1, HMGB1/2, and topoisomerase II. DFF is regulated by multiple pre‐ and post‐activation fail‐safe steps, which include the requirements for DFF45/ICAD, Hsp70, and Hsp40 proteins to mediate appropriate folding during translation to generate a potentially activatable nuclease, and the synthesis in stoichiometric excess of the inhibitors (DFF45/35; ICAD‐S/L). By contrast, Endo G resides in the mitochondrial intermembrane space in normal cells, and is released into the nucleus upon apoptotic disruption of mitochondrial membrane permeability in association with co‐activators such as apoptosis‐inducing factor (AIF). Understanding further regulatory check‐points involved in safeguarding non‐apoptotic cells against accidental activation of these nucleases remain as future challenges, as well as designing ways to selectively activate these nucleases in tumor cells.

Action of recombinant human apoptotic endonuclease G on naked DNA and chromatin substrates: Cooperation with exonuclease and DNase I

Endonuclease G (endoG) is released from mitochondria during apoptosis and is in part responsible for internucleosomal DNA cleavage. Here we report the action of the purified human recombinant form of this endonuclease on naked DNA and chromatin substrates. The addition of the protein to isolated nuclei from non-apoptotic cells first induces higher order chromatin cleavage into DNA fragments ≥ 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal (∼190 bases) and subnucleosomal (∼10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown.

Activation of the Apoptotic Endonuclease DFF40 (Caspase-activated DNase or Nuclease) OLIGOMERIZATION AND DIRECT INTERACTION WITH HISTONE H1

DNA fragmentation factor (DFF) is a heterodimeric protein composed of 45-kDa (DFF45) and 40-kDa (DFF40) subunits, a protein that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals. DFF45 is a specific molecular chaperone and an inhibitor for the nuclease activity of DFF40. Previous studies have shown that upon cleavage of DFF45 by caspase-3, the nuclease activity of DFF40 is relieved of inhibition. Here we further investigate the mechanism of DFF40 activation. We demonstrate that DFF45 can also be cleaved and inactivated by caspase-7 but not by caspase-6 and caspase-8. The cleaved DFF45 fragments dissociate from DFF40, allowing DFF40 to oligomerize to form a large functional complex that cleaves DNA by introducing double strand breaks. Histone H1 directly interacts with DFF, confers DNA binding ability to DFF, and stimulates the nuclease activity of DFF40 by increasing its K cat and decreasing itsK m .

Bulky DNA adducts in human sperm: relationship with fertility, semen quality, smoking, and environmental factors

The integrity of DNA of spermatogenic cells can be affected by endogenous and exogenous genotoxic factors. Resulting DNA damage in spermatozoa may significantly contribute to impaired fertility. Here, the 32P-postlabeling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 males, either healthy donors or patients with an impaired fertility. When all donors were analyzed, the levels of bulky DNA adducts was 1.2-fold higher in smokers than in non-smokers, but the difference was not statistically significant (P=0.054). However, a statistically significant difference existed between current smokers and never smokers among the healthy individuals (1.7-fold increase, P=0.008). No correlation between alcohol or coffee consumption and sperm DNA adducts was found. The levels of DNA adducts in sperm seemed to be unaffected by environmental and occupational factors. On the other hand, groups of healthy persons and patients with male-factor infertility differed significantly with respect to the level of bulky DNA adducts (P=0.012). A significant negative correlation between DNA adducts and sperm concentration or sperm motility existed among patients with an impaired fertility (n=93; P<0.029, r(S)=-0.225). These results suggest that DNA adducts in sperm cells can be applied as potential biomarkers in studies of human infertility.

Regulation of the human apoptotic DNase/RNase endonuclease G: involvement of Hsp70 and ATP.

Endonuclease G (EndoG) is a mitochondrial enzyme that becomes an apoptotic nuclease when released from the mitochondrial intermembrane space. EndoG will digest either DNA or RNA, but at physiological ionic strength, RNA is a much more favorable substrate as compared to chromatin. This indicates that EndoG’s major in vivo function(s) may be: (i) an apoptotic RNase, and/or (ii) an apoptotic DNase in the presence of additional co-activators. In the present study we have searched for factors that modulate the activity of human EndoG on DNA substrates. We demonstrate that EndoG forms complexes with AIF and FEN-1 but not with PCNA. Interestingly, heat shock proteins 70 interact with EndoG and are involved in the regulation of its activity. Purified Hsp70 prevented stimulation of EndoG DNase activity by other nuclear factors in the ATP-dependent manner.

Curcumin induces caspase-3-dependent apoptotic pathway but inhibits DNA fragmentation factor 40/caspase-activated DNase endonuclease in human Jurkat cells

Curcumin is a natural pigment that has been shown to induce cell death in many cancer cells; however, the death mode depends on the cell type and curcumin concentration. Here we show that, in Jurkat cells, 50 μmol/L curcumin severely lowers cell survival and induces initial stage of chromatin condensation. It also induces caspase-3, which is sufficient to cleave DNA fragmentation factor 45 [DFF45/inhibitor of caspase-activated DNase (ICAD)], the inhibitor of DFF40/CAD endonuclease. However, the release of DFF40/CAD from its inhibitor does not lead to oligonucleosomal DNA degradation in curcumin-treated cells. Moreover, curcumin treatment protects cells from UVC-induced oligonucleosomal DNA degradation. In biochemical experiments using recombinant DFF activated with caspase-3, we show that curcumin inhibits plasmid DNA and chromatin degradation although it does not prevent activation of DFF40/CAD endonuclease after its release from the inhibitor. Using DNA-binding assay, we show that curcumin does not disrupt the DNA-DFF40/CAD interaction. Instead, molecular modeling indicates that the inhibitory effect of curcumin on DFF40/CAD activity results from curcumin binding to the active center of DFF40/CAD endonuclease. [Mol Cancer Ther 2006;5(4):927–34]

Mass spectrometry-based serum proteome pattern analysis in molecular diagnostics of early stage breast cancer

BackgroundMass spectrometric analysis of the blood proteome is an emerging method of clinical proteomics. The approach exploiting multi-protein/peptide sets (fingerprints) detected by mass spectrometry that reflect overall features of a specimens proteome, termed proteome pattern analysis, have been already shown in several studies to have applicability in cancer diagnostics. We aimed to identify serum proteome patterns specific for early stage breast cancer patients using MALDI-ToF mass spectrometry.MethodsBlood samples were collected before the start of therapy in a group of 92 patients diagnosed at stages I and II of the disease, and in a group of age-matched healthy controls (104 women). Serum specimens were purified and the low-molecular-weight proteome fraction was examined using MALDI-ToF mass spectrometry after removal of albumin and other high-molecular-weight serum proteins. Protein ions registered in a mass range between 2,000 and 10,000 Da were analyzed using a new bioinformatic tool created in our group, which included modeling spectra as a sum of Gaussian bell-shaped curves.ResultsWe have identified features of serum proteome patterns that were significantly different between blood samples of healthy individuals and early stage breast cancer patients. The classifier built of three spectral components that differentiated controls and cancer patients had 83% sensitivity and 85% specificity. Spectral components (i.e., protein ions) that were the most frequent in such classifiers had approximate m/z values of 2303, 2866 and 3579 Da (a biomarker built from these three components showed 88% sensitivity and 78% specificity). Of note, we did not find a significant correlation between features of serum proteome patterns and established prognostic or predictive factors like tumor size, nodal involvement, histopathological grade, estrogen and progesterone receptor expression. In addition, we observed a significantly (p = 0.0003) increased level of osteopontin in blood of the group of cancer patients studied (however, the plasma level of osteopontin classified cancer samples with 88% sensitivity but only 28% specificity).ConclusionMALDI-ToF spectrometry of serum has an obvious potential to differentiate samples between early breast cancer patients and healthy controls. Importantly, a classifier built on MS-based serum proteome patterns outperforms available protein biomarkers analyzed in blood by immunoassays.

In Vitro Chromatin Assembly of the HIV-1 Promoter ATP-DEPENDENT POLAR REPOSITIONING OF NUCLEOSOMES BY Sp1 AND NFκB

Nuclease hypersensitive sites exist in vivo in the chromatin of the integrated human immunodeficiency virus (HIV)-1 proviral genome, in the 5′-long terminal repeat (LTR) within the promoter/enhancer region near Sp1 and NFκB binding sites. Previous studies from the Kadonaga and Jones laboratories have shown that Sp1 and NFκB can establish hypersensitive sites in a truncated form of this LTR when added before in vitro chromatin assembly with Drosophila extracts, thus facilitating subsequent transcriptional activation of a linked reporter gene upon the association of additional factors (Pazin, M. J., Sheridan, P. L., Cannon, K., Cao, Z., Keck, J. G., Kadanaga, J. T., and Jones, K. A. (1996) Genes & Dev. 10, 37–49). Here we assess the role of a full-length LTR and 1 kilobase pair of downstream flanking HIV sequences in chromatin remodeling when these transcription factors are added after chromatin assembly. Using Xenopus laevis oocyte extracts to assemble chromatin in vitro, we have confirmed that Sp1 and NFκB can indeed induce sites hypersensitive to DNase I, micrococcal nuclease, or restriction enzymes on either side of factor binding sites in chromatin but not naked DNA. We extend these earlier studies by demonstrating that the process is ATP-dependent when the factors are added after chromatin assembly and that histone H1, AP1, TBP, or Tat had no effect on hypersensitive site formation. Furthermore, we have found that nucleosomes upstream of NFκB sites are rotationally positioned prior to factor binding and that their translational frame is registered after binding NFκB. On the other hand, binding of Sp1 positions adjacent downstream nucleosome(s). We term this polar repositioning because each factor aligns nucleosomes only on one side of its binding sites. Mutational analysis and oligonucleotide competition each demonstrated that this remodeling required Sp1 and NFκB binding sites.

Ionic and cofactor requirements for the activity of the apoptotic endonuclease DFF40/CAD.

The endonuclease DFF40/CAD mediates regulated DNA fragmentation and chromatin condensation in cells undergoing apoptosis. Here we report the enzymes co-factor requirements, and demonstrate that the ionic changes that occur in apoptotic cells maximize DFF40/CAD activity. The nuclease requires Mg2+, exhibits a trace of activity in the presence of Mn2+, is not co-stimulated by Ca2+, is inhibited by Zn2+ or Cu2+, and has high activity over a rather broad pH range (7.0–8.5). The enzyme is thermally unstable, and is rapidly inactivated at 42°C. Enzyme activity is markedly affected by ionic strength. At the optimal [K+] of 50–125 mM, which is in the range of the cytoplasmic [K+] for cells undergoing apoptosis, the activity of DFF40/CAD for naked DNA cleavage is about 100-fold higher than at 0 or 200 mM [K+]. Although these ranges of ionic strength do not affect DFF40 homo-oligomer formation, at higher ionic strengths the enzyme introduces single-stranded nicks into supercoiled DNA.

Heat shock transcription factor 1 down-regulates spermatocyte-specific 70 kda heat shock protein expression prior to the induction of apoptosis in mouse testes

Expression of constitutively active heat shock transcription factor 1 (HSF1) in mouse spermatocytes induces apoptosis and leads to male infertility. We report here that prior to the onset of massive apoptosis caused by expression of active HSF1 in spermatocytes a marked reduction in spermatocyte‐specific Hsp70.2 mRNA and protein levels occurs. In addition, HSP70.2 protein relocalizes from a predominant cytoplasmic to a nuclear position in developing spermatocytes that express active HSF1. Later in the developmental stages, cells undergoing HSF1‐induced apoptosis essentially lack the HSP70.2 protein. The down‐regulation of Hsp70.2 gene expression by HSF1 is paradoxical because HSF1 is the prototypical activator of HSP genes. Furthermore, HSF1‐mediated repression neither involved a heat shock element (HSE)‐like sequence adjacent to the Hsp70.2 gene nor were Hsp70.2 promoter sequences associated directly with HSF1. Interestingly, other spermatocyte‐ and spermatid‐specific transcripts are also down‐regulated in testes of transgenic mice expressing active HSF1, suggesting involvement of a putative HSF1‐dependent block of development of spermatogenic cells. Importantly however, transcription of the Hsp70.2 gene is down‐regulated in testes of wild‐type mice subjected to a hyperthermia that induces transient activation of HSF1, indicating that the spermatocyte‐specific activity of HSF1 might misdirect a network of transcription factors required for proper regulation of Hsp70.2.