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.

A novel activation mechanism of caspase-activated DNase from Drosophila melanogaster.

Caspase-activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M(r) of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophila inhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)(2)(p20)(2) complex in gel filtration. When a Drosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD, Drosophila CAD must be cleaved by caspases to be activated.

Identification and developmental expression of inhibitor of caspase-activated DNase (ICAD) in Drosophila melanogaster.

DNA fragmentation, a hallmark of apoptosis, is regulated by a specific nuclease called caspase-activated DNase (CAD) and its inhibitor (ICAD). When cell lysates from Drosophila S2 cells were chemically denatured and the denatured proteins were removed after dialysis, the supernatant inhibited Drosophila CAD (dCAD). To identify the inhibitor, we tested recombinant DREP-1, which was previously identified using the Drosophila EST data base and found it also inhibited dCAD DNase. An antibody against DREP-1 inhibited the ICAD activity in the S2 cell extracts, confirming the identification of DREP-1 as a Drosophila homolog of ICAD (dICAD). The recombinant DREP-1/dICAD was cleaved at a specific site by human caspase 3 as well as by extracts prepared from S2 cells undergoing apoptosis. Biochemical fractionation and immunoprecipitation of dICAD from S2 cell extracts indicated that dICAD is complexed with dCAD in proliferating cells. The expression of the caspase-resistant form of dICAD/DREP-1 in a Drosophila neuronal cell line prevented the apoptotic DNA fragmentation. Northern hybridization and the immunohistochemical analyses revealed that the expression of the dICAD gene is developmentally regulated.

An approach to identify new genes in autoimmune diseases: lessons from rheumatoid arthritis.

We have searched the human genome for genes that predispose to rheumatoid arthritis using fluorescence-based microsatellite marker analysis and affected sib-pair linkage studies. A panel of 41 Japanese families, each with at least two affected siblings, was typed for genome-wide 358 polymorphic microsatellite marker loci. Three principal chromosome regions of linkage, D1S253/214, D8S556 and DX1232, have been assigned, which we call RA1, RA2 and RA3 for rheumatoid arthritis disease loci. We are now assigning the death receptor 3 as a candidate gene for RA1, and the truncated form of Dbl proto-oncogene, which does not contain the 23rd and 24th exons, as disease gene for RA3. Microsatellite marker analyses seem to be promising and new genes are now being identified by reference to sequence tag sites.

The human caspase-activated DNase gene (hCAD): genomic structure, exonic single-nucleotide polymorphisms, and a highly polymorphic dinucleotide repeat at the hCAD locus.

Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. We determined its genomic structure and identified single-nucleotide polymorphisms (SNPs) within exons 5 and 7, as well as a highly polymorphic dinucleotide repeat of (CT)m(CA)n within the 5' region of the human CAD gene (hCAD). The genomic structure of hCAD presented here, together with information concerning SNPs within the gene, as well as a highly polymorphic (CT)m(CA)n repeat fragment at the hCAD locus, may assist in the construction of genetic maps for exploring gene(s) that play pivotal roles in carcinogenesis.

Molecular cloning and characterization of human caspase-activated DNase.

Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA library. One class of cDNA encoded a protein comprising 338 amino acids, which showed a marked similarity to its murine counterpart. In vitro transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contained many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional CAD gene was localized to human chromosome 1p36.3 by fluorescent in situ hybridization. The CAD mRNA was expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlated with their ability to show DNA fragmentation during apoptosis. Overexpression of CAD potentiated DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation.

Venous drainage through bone marrow after replantation: an experimental study.

Venous drainage is vital for successful replantation, but it is not always possible to reconstruct because of missing or damaged veins. We devised an experimental model to study venous drainage through bone marrow while the new subcutaneous venous system regenerated. Adult male Wistar rats were placed into three groups. Group A rats had their hindlimbs amputated at the lower leg, but the tibia and sural and saphenous artery connections were preserved. Group B rats were prepared the same as Group A, except that a step-cut osteotomy was performed in the tibia. The bone ends were then realigned and kept in place with stainless steel wire. Group C rats were prepared the same as Group B, except that the ends of the bone were not aligned. All unoperated limbs served as controls for evaluations of blood flow. Experimental limbs were evaluated for skin colour and viability, blood flow and dye injection. Skin colour was investigated daily. Blood flow was measured postoperatively during three phases: immediate (up to 1 h), early (from 1 h to 24 h), and late (from 1 day to 7 days after operation). Survival of limbs varied in Groups A and B, while all limbs in Group C necrosed by day 7. Blood flow was returning to near control (normal) levels by day 7 in Group A and B limbs. India ink was observed in the medullary cavity at day 7. After replantation, bone marrow plays a critical role in venous drainage until the subcutaneous venous drainage system regenerates.

Presence of atypical laminin on the surface of mouse Lewis lung carcinoma cells.

We investigated the expression and distribution of laminin in Lewis lung carcinoma LL2-Lu3 cells. The microscopic immunofluorescence study of the non-permeabilized cells and blotting assay after immunoprecipitation with anti-laminin antibodies of biotinylated cell surface proteins demonstrated that LL2-Lu3 cells retained laminin on their cell surfaces. This laminin was atypical in that it lacked A chain as revealed by the immunoblot analysis. The results of the reverse transcription polymerase chain reaction method indicated that LL2-Lu3 cells contained mRNA for B1 and B2 chains, but not A chain corresponding to those of typical laminin derived from murine Engelbreth-Holm-Swarm sarcoma. A precursor form of 67 kDa laminin receptor protein was also shown to exist on the surfaces of LL2-Lu3 cells. These findings suggest that the interaction between atypical laminin and the precursor form of the 67 kDa laminin receptor protein on the cell surfaces may function in regulating cell activities such as metastasis of LL2-Lu3 cells.

Effects of catechins on the mouse tumor cell adhesion to fibronectin.

We studied the effects of 5 kinds of catechins on the adhesion of mouse lung carcinoma 3LL and melanoma B16F10 cells to the fibronectin substratum. (-)-Epicatechin gallate and (-)-epigallocatechin gallate were active in inhibiting the 3LL cell adhesion, while (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin were inactive. Gallate-containing catechins also impaired adhesion and/or spreading of B16F10 cells.

EC syndrome in a girl with paracentric inversion (7)(q22.1;q36.3).

Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome associated with a paracentric inversion of chromosome 7 in a 2-year-old Japanese girl is reported. She had sparse and light-brown hair, bilateral cleft lip and palate, fused lower incisors, a pigmented skin lesion at the neck, accessory nipples, limited extension of elbow joints and bilateral ectrodactyly of hands and feet. Cytogenetic studies demonstrated a balanced inv(7)(q22.1;q36.3) in the patient and her father. The association of EEC syndrome and inv(7) in the patient suggested a putative locus of the EEC syndrome gene either at 7q22.1 or 7q36.3, although a coincidental occurrence of the two conditions is an alternative explanation. A comparison with reported karyotypes in patients with EEC or isolated ectrodactyly favoured 7q22.1 as the locus. A normal phenotype of the father in our family might reflect reduced penetrance of the EEC syndrome or, possibly, reduced expression of a maternally-derived allele of the EEC syndrome gene through a genomic imprinting mechanism.

Diagnostic value of serum antibody to candida in an extensively burned patient.

This report gives details of an extensively burned patient who died of candidiasis. Retrospectively, by using the procedure of indirect immunoperoxidase staining, we measured the levels of antibody to candida in the stored patient's serum, collected from the time of admission to our hospital until death. Gradual increases in the antibody titres to candida were seen over the postburn period. The findings can be useful in diagnosing candidiasis.