The proteolysis of lamins, the major structural proteins of the nuclear envelope, is observed in different cells undergoing apoptosis (Zhivotovsky et al., 1997; Takahashi et al., 1996; Orth et al., 1996) and may be responsible for some of the observed nuclear changes, since inhibitors of Lamin cleavage prevent some of these changes (Lazebnik et al., 1995). An in vitro model of apoptosis has been developed in which normal nuclei, exposed to the cytosol of apoptotic cells, undergo many of the characteristic biochemical and morphological changes of nuclear apoptosis, including chromatin condensation, fragmentation and margination, internucleosomal cleavage of DNA, and proteolysis of PARP and lamins (Lazebnik et al., 1994). This cell-free system was used to characterise proteinase(s) that cleave the nuclear lamins during apoptosis. It was shown that Lamin cleavage during apoptosis requires the action of a caspase, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase caspase-3, implicating multiple caspases for apoptotic events in these cell-free extracts. Inhibition of the Lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies (Lazebnik et al., 1995).

Using cloned recombinant caspases and a cell-free model system for apoptosis (S/M extracts) the substrate specificity's of caspase-3 and caspase-6 were compared. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Caspase-6 alpha also cleaved recombinant and nuclear Lamin A at a conserved VEID¯ N sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the Lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, caspase-3 did not cleave Lamin A. (Takahashi et al., 1996; Orth et al., 1996). Under conditions where caspase-6 cleaves Lamin A, caspase-1,-3 and -7 did not, suggesting that caspase-6 may be the major lamina in cells undergoing apoptosis (Takahashi et al., 1996; Orth et al., 1996).

However it was shown recently that two different proteases are involved in the proteolysis of Lamin during apoptosis. Isolated rat thymocyte nuclei were incubated either in the presence of Ca²⁺ and Mg²⁺ or with cytosolic extract from Jurkat T lymphocytes and treated with anti-Fas antibody. Inhibitors of caspases (VADcmk, DEVDcho) were not effective in hindering Ca²⁺-induced apoptotic changes in isolated nuclei, but did prevent similar changes in nuclei treated with the cytosolic extract from apoptotic Jurkat cells. In contrast, the inhibitor of the Ca²⁺-regulated, nuclear scaffold-associated serine protease, AAPFcmk, was able to inhibit Lamin B1 breakdown, as well as chromatin cleavage in nuclei incubated in the presence of Ca²⁺ and Mg²⁺, but only partially prevented the same changes induced with cytosolic extract. These findings provide evidence for the involvement of at least two proteases in Lamin cleavage. One belonging to the caspase family which cleaves lamins and must be translocated from the cytoplasm into the nucleus. The second protease has a nuclear location and is activated by Ca²⁺ (Zhivotovsky et al., 1997). More evidence for the involvement of different caspases in Lamin cleavage comes from experiment carried out in CD95-treated HeLa cells. Here Lamin B is preferentially cleaved early in apoptosis prior to cleavage of lamins A en C and internucleosomal cleavage of DNA (Mandal et al., 1996)

The U1 small nuclear ribonucleoprotein particle is essential for splicing of precursor mRNA, an activity that depends upon both the RNA and protein components of the U1 particle. One of the U1- specific proteins that is functionally important in this splicing reaction is the 70-kDa protein (U1-70kDa). This U1- 70kDa is specifically cleaved in apoptotic cells, resulting in the generation of a 40-kDa fragment. The kinetics of this cleavage coincided with the appearance of cells with apoptotic morphology in the population, and the proportion of 40-kDa fragment observed was markedly increased in apoptotic cells that had become detached from the substratum. Although the inhibitor characteristics of the activity cleaving U1-70kDa suggest caspase-1 might be responsible, the specific caspase-1 inhibitor YVAD-CHO did not prevent cleavage, and U1-70kDa was not cleaved by purified caspase-1 in vitro (Casciola-Rosen et al., 1994). Instead purified caspase-3 cleaves U1-70 kDa at a DGPD¯ G site, similar to the reaction observed in apoptotic cells. I lysates from apoptotic cells, the cleavage of U1-70 kDa is potently inhibited by Ac-DEVD.CHO but not by Ac-YVAD.CHO, strongly suggesting that caspase-3 or possibly caspase-7 is the major caspase responsible for its cleavage in apoptosis. Cleavage of U1-70 kDa separates the RNA binding domain from the distal arginine-rich region of the molecule, which may have a dominant-negative effect on splicing; such inhibition of splicing would block cellular repair pathways dependent on new mRNA synthesis (Casciola Rosen et al., 1994).

PARP is possibly the best characterised proteolytic substrate of caspases, being cleaved in the execution phase of apoptosis in many systems, including thymocytes, HL-60c cells and breast cancer cell lines Intact PARP (116 kDa) is cleaved to 24 kDa and 89 kDa fragments, representing the N-terminal DNA binding domain and the C-terminal catalytic domain of the enzyme respectively.

PARP has a very high activity and, if massively activated by the chromatin fragmentation that frequently occurs in cells undergoing apoptosis, it could deplete cellular ATP stores. This would endanger the apoptotic pathway, since the formation of apoptotic bodies requires ATP (Schwartzmann and Cidlowski, 1993). Inactivating PARP conserves the cellular NAD⁺ and ATP normally required for PARP activity, thereby enabling the ATP to be utilised for the execution of apoptosis and avoid inflammation (Takahashi and Earnshaw, 1996). Cleavage of PARP might protect the apoptotic cell in several ways (Earnshaw, 1995). First, the cleavage separates the DNA-binding and catalytic domains. This renders the catalytic domain insensitive to DNA cleavage (Kaufmann et al., 1993). Second, the N-terminal DNA binding fragment can act as a dominant negative inhibitor of intact PARP by competing for free DNA ends (Molinete and et al. 1993). So cleavage of PARP may also interfere with its key homeostatic function as a DNA repair enzyme (Kaufmann et al., 1993). Third, the cleavage occurs in the middle of the PARP bipartite nuclear localisation signal (Schreiber et al., 1992). Thus, any of the 85 kDa C-terminal fragment present in the cytoplasm will be unable to enter the nucleus. This fragment retains enzymatic activity and may stimulate apoptosis by modifying target cytoplasmatic polypeptides.

PARP is cleaved at the sequence DEVD¯ G by a protease activity resembling ICE (prICE), but not by ICE itself (Lazebnik et al., 1994). In vitro, many caspases, including caspase-2,-4,-6,-7,-8,-9 and -10, when added at high concentrations, can cleave PARP or DEVD-AMC (Gu et al., 1995). The physiological significance of this cleavage of PARP by these caspase is still under investigation. It appears that caspase-3 and caspase-7 are primarily responsible for PARP cleavage during apoptosis

Interestingly it was reported that during necrosis, PARP is degraded differently from that observed during apoptosis. While apoptotic HL-60 cells exhibit only the signature 89 kDa fragment of PARP, necrosis of these cells is accompanied by formation of major fragments at approximately 89 and 50 kDa and minor fragments at approximately 40 and 35 kDa. The necrosis-specific degradation of PARP was coincident with other changes detected by flow cytometric analysis, but earlier than the extensive degradation of DNA (Shah et al., 1996).

Poly-ADP-ribosylation is implicated in many fundamental processes, like DNA repair, chromatin stability, cell proliferation, and cell death. To elucidate the biological function of protein(ADP-ribosyl) transferase (ADPRT = PARP), a chromatin-associated enzyme which, in the presence of DNA breaks, transfers ADP-ribose from NAD⁺ to nuclear proteins, in vivo the gene was inactivated in the mouse germ line. Mice homozygous for the PARP mutation are healthy and fertile. Analysis of mutant tissues and fibroblasts isolated from mutant foetuses revealed the absence of PARP enzymatic activity and poly(ADP-ribose), implying that no poly-ADP-ribosylated proteins are present. Mutant embryonic fibroblasts were able to efficiently repair DNA damaged by UV and alkylating agents (Wang et al., 1995).

This was confirmed by comparing the susceptibility of cells from wild- type mice and PARP^-/- mice to several inducers of apoptosis. Neither the susceptibility of hepatocytes towards FAS or TNF- mediated apoptosis nor the activation of PARP-cleaving caspases was modified in PARP^-/- liver cells. Thymocytes with either genotype exhibited similar sensitivity to treatments with ceramide, dexamethasone, or etoposide. The sensitivity of primary neurons towards apoptosis induced by staurosporine, colchicine, potassium withdrawal, peroxynitrite, or the neurotoxin MPP⁺ was also unaltered suggesting that neither activation nor cleavage of PARP has a causal role in apoptotic cell death of primary, non-transformed cells (Leist et al., 1997). So although PARP is often a valuable indicator of apoptosis, its biological relevance, if any, is unclear.

The specific cleavage of the heteronuclear ribonucleoproteins (hnRNPs) C1 and C2, abundant nuclear proteins thought to be involved in RNA splicing, in apoptotic cells induced to undergo apoptosis by a variety of stimuli, including ionising radiation, etoposide, and ceramide was reported. No cleavage was observed in cells that are resistant to apoptosis induced by ionising radiation. Protease inhibitor data implicate the involvement of caspases in the cleavage of hnRNP C. Using recombinant caspases and purified hnRNP C proteins in vitro, is was shown that the hnRNP C proteins are cleaved by caspase-3,-7 and, to a lesser extent, by caspase-6, but not by caspase-1,-2,-4 or the cytotoxic T-cell protease granzyme B (Waterhouse et al., 1996).

DNA-PK, an enzyme involved in DNA double-strand-break repair, possesses a 460 kDa catalytic subunit (DNA-PK_CS) and a DNA binding component Ku, which is a heterodimer of 70 and 80 kDa subunits. It was demonstrated that DNA-PK_CS is an excellent substrates for caspase-3, with cleavage occurring at a site that is highly similar to the cleavage site within PARP. The fragments generated from isolated protein substrates by caspase-3 are identical to those observed in intact apoptotic cells, in apoptotic cell extracts, and in normal cell extracts to which caspase-3 has been added. Like PARP, cleavage of DNA-PK_CS in apoptotic cell extracts is abolished by nanomolar concentrations of Ac-DEVD-CHO and micromolar amounts of Ac-YVAD-CHO, confirming the involvement of caspase-3 - like activity. Degradation of DNA-PK_CS should lead to a decrease in the DNA repair capacity of the cell, so abolishing its key homeostatic function and facilitating the characteristic DNA degradation associated with apoptosis (Casciola-Rosen et al., 1996).

From HeLa cytosol a protein was purified that induces DNA fragmentation in coincubated nuclei after it is activated by caspase-3. This protein, designated DNA Fragmentation Factor (DFF), is a heterodimer of 40 kDa and 45 kDa subunits. The amino acid sequence of the 45 kDa subunit, determined from its cDNA sequence, reveals it to be a novel protein. Caspase-3 cleaves the 45 kDa subunit at two sites to generate an active factor that produces DNA fragmentation without further requirement for caspase-3 or other cytosolic proteins. In cells undergoing apoptosis, the 45 kDa subunit is cleaved in the same pattern as it is cleaved by caspase-3 in vitro. These data delineate a direct signal transduction pathway during apoptosis: caspase-3 to DFF to DNA fragmentation (Liu et al., 1997)