Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN.

The most frequently expressed drug resistance genes, MDR1 and MRP1, occur in human tumors with mutant p53. However, it was unknown if mutant p53 transcriptionally regulated both MDR1 and MRP1. We demonstrated that mutant p53 did not activate either the MRP1 promoter or the endogenous gene. In contrast, mutant p53 strongly up-regulated the MDR1 promoter and expression of the endogenous MDR1 gene. Notably, cells that expressed either a transcriptionally inactive mutant p53 or the empty vector showed no endogenous MDR1 up-regulation. Transcriptional activation of the MDR1 promoter by mutant p53 required an Ets binding site, and mutant p53 and Ets-1 synergistically activated MDR1 transcription. Biochemical analysis revealed that Ets-1 interacted exclusively with mutant p53s in vivo but not with wild-type p53. These findings are the first to demonstrate the induction of endogenous MDR1 by mutant p53 and provide insight into the mechanism.The emergence of drug resistance poses a major obstacle to the success of cancer chemotherapy. Tumor cells acquire drug resistance via many routes including alterations in transport, drug targets, metabolism, and/or genes regulating cell survival. The most common alterations in drug transport are increased expression of MDR1 1 (the gene product is P-glycoprotein (1, 2)) and the multidrug resistance-associated protein (MRP1) (3, 4). Both are energy-dependent anticancer drug efflux pumps and play critical roles in the response to chemotherapeutic drugs (e.g. vinca alkaloids, taxanes, and epipodophyllotoxins). Further, both MDR1 and MRP1 are expressed in colon tumors that frequently express mutant (MT) forms of p53 (5, 6) and are intractable to chemotherapy. Notably, we have shown directly that MDR1 in colon tumors is normally repressed by wild-type (wt) p53 (7). In an analogous fashion Wang and Beck (9) as well as Sullivan et al. (8) have shown that wt p53 represses MRP1. Many clinical studies show that MT p53 expression is associated with increased MDR1 and/or MRP1 expression (5, 10, 11). These findings are fully consistent with a loss of p53 repression leading to MDR1 or MRP1 up-regulation. However, it is just as likely that these genes could be up-regulated by the "gain-offunction" activity of MT p53s (12-14).p53 deletion or mutation is one of the most frequent alterations in human malignancy and is clearly a critical step in the progression of colorectal cancer (15). Close to 90% of the p53 mutations in human tumors results in a disruption of the DNA binding domain. This not only disrupts transrepression and sequence-specific transactivation but also confers a gain-offunction activity that was first demonstrated for many MT p53s as acquiring the ability to induce tumors (13). This property was associated with the capability of these MT p53s to stimulate the expression of an alternate set of endogenous genes (13, 14, 16) that could potentially promote tumor progression and impair therapeutic response. However, although c-myc has unequivocally been demonstrated to be an endogenous t...

Although it has been reported that cyclin L1␣ and L2␣ proteins interact with CDK11 p110 , the nature of the cyclin L transcripts, the formation of complexes between the five cyclin L and the three CDK11 protein isoforms, and the influence of these complexes on splicing have not been thoroughly investigated. Here we report that cyclin L1 and L2 genes generate 14 mRNA variants encoding six cyclin L proteins, one of which has not been described previously. Using cyclin L gene-specific antibodies, we demonstrate expression of multiple endogenous cyclin L proteins in human cell lines and mouse tissues. Moreover, we characterize interactions between CDK11 p110 , mitosis-specific CDK11 p58 , and apoptosis-specific CDK11 p46 with both cyclin L␣ and -␤ proteins and the co-elution of these proteins following size exclusion chromatography. We further establish that CDK11 p110 and associated cyclin L␣/␤ proteins localize to splicing factor compartments and nucleoplasm and interact with serine/arginine-rich proteins. Importantly, we also determine the effect of CDK11-cyclin L complexes on pre-mRNA splicing. Preincubation of nuclear extracts with purified cyclin L␣ and -␤ isoforms depletes the extract of in vitro splicing activity. Ectopic expression of cyclin L1␣, L1␤, L2␣, or L2␤ or active CDK11 p110 individually enhances intracellular intron splicing activity, whereas expression of CDK11 p58/p46 or kinase-dead CDK11 p110 represses splicing activity. Finally, we demonstrate that expression of cyclins L␣ and -␤ and CDK11 p110 strongly and differentially affects alternative splicing in vivo. Together, these data establish that CDK11 p110 interacts physically and functionally with cyclin L␣ and -␤ isoforms and SR proteins to regulate splicing.It has become apparent over the past decade that several cyclin-dependent kinases (CDKs) 4 and their cyclin regulatory partners participate in regulating mRNA production (1). Thus far, CDK7, CDK8, and CDK9 functions are ascribed to transcriptional initiation and elongation, and CDK12 (CrkRS) and CDK13 (CDC2L5) functions are related to pre-mRNA splicing (2-4). Interestingly, CDK11 p110 plays roles in both transcription and splicing, suggesting that this CDK may link the two processes (5, 6). In addition, the CDK11 p110 partner proteins cyclins L1 and L2 also influence splicing (7,8). Two distinct genes, Cdc2L1 and Cdc2L2 (acronym for Cell division control 2 Like), encode the human p110 and p58 PITSLRE protein kinases (9 -12). These kinases were renamed CDK11 p110 and CDK11 p58 when cyclins L1 and L2 were identified as regulatory subunits of CDK11 p110 (13). Expression of the CDK11 p110 isoforms is ubiquitous and constant throughout the cell cycle (11). In contrast, CDK11 p58 is expressed and functions specifically in G 2 /M via an internal ribosome entry site (IRES) located within the CDK11 p110 mRNA (14 -17). During apoptosis, a third isoform, CDK11 p46 , is generated by caspase-dependent cleavage of CDK11 p110 and CDK11 p58 , leaving the catalytic domain intact (18,19).A role for CDK11 p...

Chromatin structure is influenced by histone modification, and this may help direct chromatin behavior to facilitate transcription, DNA replication, and DNA repair. Chromatin condensation and DNA fragmentation are the classic nuclear features but remain poorly characterized. It is highly probable that nucleosomal structure must be altered to allow these features to become apparent, but data to support this construct are lacking. We report here that in response to apoptotic signals from a death receptor (CD95 and tumor necrosis factor-␣) or mitochondrial (staurosporine) apoptotic stimulus, the core nucleosomal histones H2A, H2B, H3, and H4 become separated from DNA during apoptosis in Jurkat and HeLa cells and are consequently detectable in the cell lysate prepared using a non-ionic detergent. The timing of this histone release from DNA correlates well with the progression of apoptosis. We also show expression of a caspase cleavageresistant form of ICAD (ICAD-DM) in Jurkat and HeLa cells abolished DNA fragmentation and also dramatically reduced histone release in apoptotic cells. However, we demonstrate that apoptotic histone release is not an inevitable consequence of CAD/DFF-40-mediated DNA destruction as DNA fragmentation but not histone release occurs efficiently in tumor necrosis factor-␣-and etoposide-treated NIH3T3 cells. Furthermore, in an in vitro apoptotic assay, incubation of apoptotic Jurkat cellular extract with non-apoptotic Jurkat nuclei led to nuclear DNA fragmentation without obvious histone release. Taken together, these data demonstrate that CAD/DFF-40 functions indirectly in mediating nucleosomal destruction during apoptosis.

Cycloheximide (CHX) can contribute to apoptotic processes, either in conjunction with another agent (e.g. tumor necrosis factor-␣) or on its own. However, the basis of this CHX-induced apoptosis has not been clearly established. In this study, the molecular mechanisms of CHX-induced cell death were examined in two different human T-cell lines. In T-cells undergoing CHX-induced apoptosis (Jurkat), but not in T-cells resistant to the effects of CHX (CEM C7), caspase-8 and caspase-3 were activated. However, the Fas ligand was not expressed in Jurkat cells either before or after treatment with CHX, suggesting that the activation of these caspases does not involve the Fas receptor. To determine whether CHXinduced apoptosis was mediated by a Fas-associated death domain (FADD)-dependent mechanism, a FADD-DN protein was expressed in cells prior to CHX treatment. Its expression effectively inhibited CHX-induced cell death, suggesting that CHX-mediated apoptosis primarily involves a FADD-dependent mechanism. Since CHX treatment did not result in the induction of Fas or FasL, and neutralizing anti-Fas and anti-tumor necrosis factor receptor-1 antibodies did not block CHXmediated apoptosis, these results may also indicate that FADD functions in a receptor-independent manner. Surprisingly, death effector filaments containing FADD and caspase-8 were observed during CHX treatment of Jurkat, Jurkat-FADD-DN, and CEM C7 cells, suggesting that their formation may be necessary, but not sufficient, for cell death.The apoptotic process is now known to involve the well orchestrated interactions of cell death receptors, death receptor adaptors, caspases, and Bcl-2 family members (1-6). Although a number of stimuli have been reported to result in the upregulation of the Fas receptor and its ligand (e.g. UV, c-Myc, and certain chemotherapeutic drugs), there are many other stimuli for which the mechanism responsible for their action is still unknown (7-10). An example of the latter is the ability of cycloheximide (CHX) 1 to either promote or inhibit apoptosis in divergent cell types and in response to varying death stimuli (11)(12)(13)(14)(15)(16). A large body of evidence has shown that CHX can potentiate, and in some cases (e.g. TNF␣ stimulation and staurosporine) be necessary for, the apoptotic effects of certain death stimuli (12)(13)(14)16 The ability of CHX to induce cell death varies considerably from one cell line to another, suggesting that the continuous synthesis of a regulatory protein that blocks apoptosis is required for the normal growth of these CHX-sensitive cell lines (12, 13). Sensitivity to CHX is not necessarily determined by cell type alone since cell lines from the same tissue and stage of development (e.g. Jurkat and CEM C7 T-cells) can be affected in very different ways. Furthermore, although cell death triggered by cell-surface receptors (e.g. Fas, DR3, and TNF receptor-1) requires an adaptor protein such as FADD to promote an apoptotic signal, cell death triggered by other stimuli (e.g. E1A, c-Myc, and Adriamyci...