Induced apoptosis in the prevention of colorectal cancer by non-steroidal anti-inflammatory drugs

Epidemiological, clinical and animal studies indicate non-steroidal anti-inflammatory drugs (NSAIDs) to be chemopreventive for colorectal cancer. The best established target for NSAIDs are the two isoforms of cyclooxygenase (COX), a key enzyme in the biosynthesis of prostaglandins. Recent investigations using human colorectal tumor cell lines have focused on the cellular and molecular mechanisms potentially underlying the chemopreventive effect of NSAIDs. These studies have used traditional NSAIDs and their metabolites which either do not inhibit COX, are non-selective for the COX isoforms or selectively inhibit COX-1 over COX-2, and recently developed NSAIDs that are highly selective for COX-2. In vitro, apoptosis is the dominant anti-proliferative effect of each of these classes of NSAID and sensitivity to NSAID-induced apoptosis increases with the malignant potential of the tumor cells. Limited in vivo evidence backs up these findings. Cell cycle arrest also contributes to the in vitro growth inhibitory effect of traditional NSAIDs. The induction of apoptosis by NSAIDs may result from the inhibition of the COX isoforms but other as yet undefined paths to NSAID-induced apoptosis clearly exist. A member of each class of NSAID is under trial as a chemopreventive agent for colorectal cancer.     

Modified approach for apoptosis detection reveals changes in apoptotic processes in the seminoma-associated tissue

Apoptosis morphology (DNA condensation) and internucleosomal DNA cleavage (TdT assay) were measured simultaneously on double fluorescence labeled testis tumor sections, employing conventional immunofluores cence microscopy. Six different apoptosis indices (AI) were determined based either solely on morphological or biochemical criteria, or on a combination of both processes. Measurements were performed in metastasized and non-metastasized seminoma, and in histological regions located distantly and associated with the tumor. Preliminary results on 19 histologies revealed that up to 66% of apoptotic cells were not detected, depending on the method used for apoptosis detection. Besides, no changes of solely morphologically defined AI was found in the different histological regions. By contrast, significant changes (p < 0.0004) in the different histological regions were detected when measuring AIs, e.g., defined by DNA fragmentation occuring without DNA condensation in apoptotic cells. Those changes were not detected in metastasized seminoma. These data, for the first time allow a comparison of two widely used approaches for apoptosis detection. Furthermore, the results reveal differences in apoptotic processes in tissue associated with non-metastasized seminoma detectable by a modified evaluated TdT assay but not by morphological changes, although this TdT method fails to show the total amount of apoptotic cells.     

Elimination of extrachromosomal c-myc genes by hydroxyurea induces apoptosis

Hydroxyurea (HU) increases extrachromosomal DNA elimination in tumor cell lines. The c-myc oncogene is one of the many relevant amplified genes contained within the extrachromosomal DNA compartment. Spontaneous loss of amplified copies of c-myc induces terminal differentiation and apoptosis in the human HL-60 leukemia cell lines. In the present study, we evaluate HU's ability to induce apoptosis by eliminating extrachromosomally located c-myc oncogene in human tumor cell lines. The consequences of eliminating extrachromosomal DNA by HU were explored in two different cell lines using the TdT assay and acridine orange/ethidium bromide labeling. COLO 320 clone 3 and COLO 320 clone 21 cell lines contain the same number of amplified copies of c-myc oncogene, but located respectively on extrachromosomal DNA, and intrachromosomally in homogeneously staining regions. HU induced apoptosis in the COLO 320 clone 3 cell line by a time and concentration dependent mechanism but could not induce apoptosis in the COLO 320 clone 21 cell line. These results suggested that HU-induced apoptosis in COLO 320 cell lines depends on elimination of extrachromosomal amplified copies of the c-myc oncogene. The ability of HU to eliminate extrachromosomally amplified copies of the c-myc oncogene and to induce apoptosis should be considered when targeting malignancies with amplification of the c-myc oncogene in an extrachromosomal site.     

Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration

Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.     

Analysis of a novel mechanism of neuronal toxicity produced by an apolipoprotein E-derived peptide

The apolipoprotein E (apoE)-derived peptide (141-155)2 has a neurotoxic effect, implying that apoE itself could be a source of toxicity in Alzheimer's disease brain. We characterized the toxicity of this peptide on superior cervical ganglion (SCG) neurons and compared the death with the apoptotic death that occurs after nerve growth factor (NGF) deprivation in these cells. A dose of 10 microM apoE (141-155)2 resulted in the death of approximately 50% of the neurons within 24 h. Nuclear condensation and DNA fragmentation preceded the death. However, most inhibitors of NGF deprivation-induced death, including the caspase inhibitor Boc-aspartyl(O-methyl)fluoromethyl ketone and genetic deletion of bax-/-, had no effect on the toxicity. Inclusion of depolarizing levels of potassium did block the toxicity. Receptor-associated peptide (RAP), an antagonist for apoE receptors, did not protect cells in either SCG or hippocampal cultures. In addition, RAP had no effect on internalization of the apoE peptide. These data support the observation that apoE (141-155)2 is neurotoxic but suggest that the neurotoxicity is distinct from classical apoptosis or necrosis. Furthermore, these results indicate that the toxic effect may occur independently of members of the low-density lipoprotein receptor gene family.     

Increased expression of rat synuclein in the substantia nigra pars compacta identified by mRNA differential display in a model of developmental target injury

Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.     

Mitochondrial Dysfunction in the Pathogenesis of Necrotic and Apoptotic Cell Death

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca²⁺ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca²⁺, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP.     

Lack of evidence for an immunosuppressive role for MUC1

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1- and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells. Received: 20 August 1998 / Accepted: 3 December 1998