Apoptosis, a morphologically defined form of physiological cell death, is implemented by a death machinery whose executionary arm is a family of cysteine proteases called caspases. These death proteases are part of a proteolytic caspase cascade that is activated by diverse apoptotic stimuli from outside and inside of the cell. The cell death machinery is evolutionarily conserved and composed of caspases and their regulatory components that include activators and repressors. These key components of the death machinery are linked to signaling pathways that are activated by either ligation of death receptors expressed at the cell surface or intracellular death signals. Caspases are normally present in the cell as proenzymes that require limited proteolysis for activation of enzymatic activity. Recent studies suggest that the basic mechanism of caspase activation is conserved in evolution. Binding of initiator caspase precursors to activator molecules appears to promote procaspase oligomerization and autoactivation. Enzymatic activation of initiator caspases leads to proteolytic activation of downstream (effector) caspases and cleavage of a number of vital proteins, resulting in the orderly demise and removal of the cell.

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-X L , an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-X L was mediated through both its CED-4-like domain and the Cterminal domain containing WD-40 repeats. Expression of Bcl-X L inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-X L purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-X L failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-X L regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-X L associates with caspase-9 and Apaf-1, and show that Bcl-X L inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

Mammary gland involution is a physiological process in which the entire organ is remodeled through the process of apoptosis. Apoptosis of secretory alveolar cells is initiated at the time of weaning, followed by the collapse and disappearance of the entire lobuloalveolar compartment. While apoptotic figures were rare in mammary epithelium of lactating mice, their number increased after weaning and reached a maximum on day 3 of involution. Active cell death continued until day 5 after weaning and only little parenchyma remained on day 8, when remodeling of the gland was completed. Bax mRNA levels increased during the first day of involution independent of the presence or absence of p53. Bax protein was detected in an increasing number of cells after weaning, peaking at day 3 and decreasing thereafter. Low levels of bcl-x mRNA and protein were present during lactation, followed by a sharp increase during the first 2 days of involution. The bcl-xS splice variant of bcl-x can promote cell death, and bcl-xL has a protective function in cell culture. The ratio of bcl-xS versus bcl-xL remained stable in the virgin, pregnant and lactating gland. However, during the first 2 days of involution, bcl-xS expression increased six-fold more than bcl-xL. To further evaluate the role of Bcl-xS which was less abundant in the mammary cells than Bcl-xL, cotransfection studies were performed in cell culture. They confirmed that Bcl-xS protein can facilitate apoptosis even when Bcl-xL is present in excess. These findings point to a significant role for Bax and Bcl-xS in the regulation of apoptosis of secretory alveolar cells during involution.

Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly speci®c EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-X L , a member the Bcl-2 family which suppresses apoptosis, were speci®cally inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-a and epidermal growth factor (EGF), increased the expression of Bcl-X L in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-X L in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-X L plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to llpl5 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, llpl5 contains >20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295-to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were >4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on llpl5. refs. 5 and 6) and the closely linked H19 (5, 7), are imprinted, i.e., show parental origin-specific gene expression in normalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.development. Furthermore, IGF2 shows loss of imprinting in embryonal tumors (5,6,8,9).The simplest hypothesis is that a single gene accounts for BWS and embryonal tumors and that balanced germ-line chromosomal rearrangements from BWS patients interrupt and, therefore, define this gene. Sait et al. (10) indirectly mapped by pulse field gel electrophoresis (PFGE) three such BWS breakpoints to a 675-kb region of ilpiS and >275 kb centromeric to IGF2. However, this distance is tentative as it was derived from the sum of several PFGE fragments, one of which was inferred from other larger overlapping PFGE fragments. Furthermore, two BWS breakpoints lie at an undetermined distance centromeric to these PFGE fragments (11). In these mapping studies, only one breakpoint has been isolated (10), and thus the precise physical relationship among them is unknown. Furthermore, indirect mapping by PFGE is limited by the large size of the fragments. Finally, the relationship between any of these breakpoints and a tumor suppressor gene on llpiS has not been determined.We cloned the region of ...

The DNA fragmentation factor (DFF) is composed of two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD).

The majority of breast cancer patients succumb to metastatic disease. We summarize published and recent research concerning the nm23 gene in breast cancer metastasis. In a murine developmental study, nm23 expression increased with the functional differentiation of the mammary gland in nulliparous and pregnant animals. In human breast cancer, five studies have now demonstrated a significant association between reduced nm23 expression, at the RNA or protein levels, and aggressive tumor behavior. Nm23-negative tumor cells have been observed in comedo ductal carcinoma in situ lesions in two independent studies, indicating that decreases in nm23 expression begin prior to actual histologically identifiable invasion. Transfection studies, in which human nm23-H1 cDNA was expressed in the metastatic human MDA-MB-435 breast carcinoma cell line, indicate that nm23-H1 suppresses in vivo metastatic potential by 50-90%. Finally, our data in melanoma and breast carcinoma transfection systems suggest that the biochemical mechanism of nm23 suppressive activity is likely not due to its nucleoside diphosphate kinase activity, association with GAP proteins, or secretion from cells.