The mechanisms of apoptosis in biology and medicine: a new focus for ophthalmology.

Defects in apoptosis (programmed cell death) have recently emerged as being closely involved in the pathogenesis of most ocular diseases and, therefore, apoptosis is now a topic of exponential interest in ophthalmology. This review summarizes recent works on mechanisms of apoptosis, from its initiation and modulation to the switching-on of its execution machinery. Interactions of cell death with cell division programs to orchestrate ontogenesis, aging, and adult life and their alterations in human diseases are pointed out. Two main apoptotic signaling pathways are identified: a death receptor-dependent (extrinsic) pathway and a mitochondrion-dependent (intrinsic) pathway. Mitochondrion harbors both antiapoptotic (Bcl-2, Bcl-XL) and apoptotic factors (Smac/Diablo, Apaf-1, cytochrome c). Its permeability transition pore (mPTP) is the main trigger of cell suicide. The process of mPTP opening, in association with extrusion to cytoplasm of a variety of apoptotic factors, is shown. Cytochrome c is one of these apoptotic factors. When expelled to cytoplasm, this double-faced respiratory chain component assembles with two other modules, Apaf-1 and procaspase 9, to form a protein complex--the apoptosome--that starts apoptosis execution. Another respiratory chain component, the CoQ10, is believed to counteract mPTP opening. What makes apoptosis particularly exciting for medicine is that its dysfunctions play a central role in the pathogenesis of several human diseases. For instance, excesses of apoptosis lead to cell loss that accompanies neurodegenerative diseases, whereas genetically determined defects of apoptosis lead to the deregulated cell proliferation typical of cancer. A variety of ophthalmologic diseases, such as post-keratectomy haze, corneal lesions, cataract, glaucoma, senile maculopathies, and genetic ocular pathologies, that underlie apoptosis dysfunctions are treated in detail in the other reviews of this issue.

Apoptosis is associated with modifications of bcl-2 mRNA AU-binding proteins.

The expression of genes requiring finely tuned control is regulated by a posttranscriptional mechanism involving mRNA A + U-rich elements (AREs) cooperating with ARE-binding proteins (AUBPs) in modulation of mRNA stability. We reported previously that an ARE in the bcl-2 mRNA 3'-untranslated region (3'-UTR) had destabilizing activity and was involved in bcl-2 downregulation during apoptosis in vitro. Here we demonstrate that the bcl-2 ARE complexes with a number of specific AUBPs, whose pattern undergoes changes following application of apoptotic stimuli. The caspase inhibitor Z-VAD-fmk strongly attenuates both bcl-2 mRNA decay and bcl-2 AUBP pattern changes elicited by apoptotic stimuli, indicating the involvement of bcl-2 AUBPs in bcl-2 mRNA stability control.

Prevention of corneal keratocyte apoptosis after argon fluoride excimer laser irradiation with the free radical scavenger ubiquinone Q10.

PURPOSE: To assess in vitro the potential of the free radical scavenger ubiquinone Q10 in preventing keratocyte apoptosis after argon fluoride (ArF) excimer laser irradiation. METHODS: Cultured rabbit keratocytes were irradiated at very low single-pulse laser fluences. The cumulative effects generated by three total fluence doses between 12 and 45 mJ/cm2, representative of single-pulse subablative doses during photorefractive keratectomy (PRK) in humans, were evaluated. We employed the following parameters to compare pretreated (10 microM ubiquinone Q10) and untreated samples: 1) number and morphology of living cells by Trypan blue test and ultramicroscopy, respectively; 2) level of free-radical formation assessed by malonaldehyde quantitation; 3) cellular energy level evaluated by ATP assay. RESULTS: Excimer laser irradiation kills cultured keratocytes by inducing apoptosis. The effect increases with the cumulative fluence dose. In the samples pretreated with ubiquinone Q10 there were significantly fewer cumulative apoptotic events than in the untreated ones. Quantitative analysis of malonaldehyde cellular levels suggested this protective action of ubiquinone Q10 was connected with its ability to scavenge laser-generated free radicals. ATP assay also confirmed that it raised cellular energy levels. CONCLUSIONS: The treatment of corneal keratocytes with relatively low concentrations of ubiquinone Q10 can prevent apoptosis after ArF excimer laser irradiation. If these findings are confirmed on human keratocytes this treatment could be usefully exploited in the PRK surgical procedure. That might lead to a reduction in the occurrence of haze and curvature regression triggered by programmed cell death.

Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis.

A rat fibroblastic cell line (rat-1/myc-ERtrade mark) was treated with different concentration of Antimycin A, a metabolic poison that affects mitochondrial respiratory chain complex III. The modes of cell death were analyzed by time-lapse videomicroscopy, in situ end-labeling (ISEL) technique, and ultrastructural analysis. Intracellular ATP levels were also measured in order to detect whether the energetic stores were determinant for the type of cell death. It was found that while apoptosis was the prevalent cell death in the fibroblasts treated with low doses, 100 or 200 microM Antimycin A, a new type of cell demise that shared dynamic, molecular, and morphological features with both apoptosis and necrosis represents the most common cell death when the cells were exposed to high doses, 300 or 400 microM, of the hypoxic stimulus. This new type of cell death has been chimerically termed aponecrosis. The inhibition of caspase 3, an enzyme critical for the apoptotic DNA degradation, caused a clear shift from aponecrosis to necrosis in the cell culture, suggesting that this new type of cell death could account for an incomplete execution of the apoptotic program and the following degeneration in necrosis. After being treated with higher doses, i.e., 1000 microM Antimycin A, almost all of the cells died by true necrosis. The analysis of the cellular energetic stores showed that the levels of ATP were a primary determinant in directing toward active cell death (apoptosis), aponecrosis, or necrosis. We conclude that chemically induced hypoxia produces different types of cell death depending on the intensity of the insult and on the ATP availability of the cell, and that the classic apoptosis and necrosis may represent only two extremes of a continuum of intermediate forms of cell demise.

Induction of apoptosis and mitosis inhibition by degraded DNA lipotransfection mimicking genotoxic drug effects.

Genotoxic damage induces cell cycle arrest and/or apoptosis by activation of p53 oncosuppressor protein. A number of anticancer drugs are genotoxic and their damaging effect upon cells is mediated by this mechanism. Microinjection of defined DNA species directly into nucleus has been reported previously to activate p53 and inhibit cell cycle. Here, we demonstrate that simple addition of heterogeneous degraded DNA to cultured cells (Rat-1 fibroblasts) in combination with lipotransfecting agent DOTAP leads to apoptosis induction and mitosis inhibition by a molecular mechanism which mimics that of the cellular response to genotoxic anticancer agents. Indeed, both cellular effects induced by lipotransfected degraded DNA (essentially, heterogeneous small DNA fragments) are associated to p53 activation and modulated by two apoptosis-related genes, such as bcl-2 and c-myc, which also modulate the apoptotic threshold to anticancer agents. Here we raise the hypothesis of exogenous DNA segment lipotransfection as possible new tool for anticancer therapy.

17beta-estradiol induces apoptosis in the preosteoclastic FLG 29.1 cell line.

Although compelling data have demonstrated the effectiveness of estrogen replacement therapy for the treatment of accelerated bone loss in postmenopausal osteoporosis and ovariectomized animals, the mechanisms by which estrogens reduce bone resorption remain to be elucidated. To address this issue, in the present study we investigated whether estrogens were able to induce programmed cell death or apoptosis in osteoclast precursors. To this purpose, a preosteoclastic cell line (FLG 29.1) was cultured in the absence or presence of nanomolar concentrations of 17beta-estradiol (17betaE2). Using time-lapse videomicroscopy, it was shown that 17betaE2 induced FLG 29.1 cell apoptosis in a dose- and time-dependent manner. Furthermore, a significant increase in the activity of caspase 3 enzyme and in the number of nuclei undergoing DNA fragmentation was observed in FLG 29.1 cells treated with 17betaE2 compared to untreated cells. Finally, transmission electron microscopy of the treated cells showed typical apoptotic morphology. These data indicate that 17betaE2 is able to promote in vitro apoptosis in preosteoclastic cells and suggest that estrogenic molecules may exert in vivo a direct role in negatively modulating the pool of undifferentiated bone marrow cells capable ultimately of maturing into osteoclasts.

[Morphologic and biochemical kinetics of the pathogenesis of acute lung injury by endotoxin in rats]. / Cinetica morfologica e biochimica della patogenesi del danno polmonare acuto da endotossina nel ratto.

BACKGROUND AND AIM: To highlight the intervention sequence of cells and their products (RO degree and NO) involved in the pathogenesis of lung injury caused by the instillation of endotoxin in rats. EXPERIMENTAL DESIGN: An experimental comparative study in rats. MATERIALS AND METHODS: The experiments were performed using intratracheal instillation of endotoxin in rats (5 mg/kg in 0.125 ml of saline solution). Untreated rats or those instilled with saline solution alone formed the control group. All animals were sacrificed 12, 24 and 48 hours after instillation and the following studies were performed on both lungs: 1) morphological study (optical and electronic); 2) assay of lung MDA; 3) NADPH-diaphorase evaluation using a histochemical method. RESULTS: Lung damage evolves gradually over 48 hours. After the first 12 hours, neutrophil granulocytes were present in the lung capillaries together with monocytes; monocytes were also present in the interstitium. During the following hours, monocytes differentiated into macrophages and, once activated, the granulocytes passed into the interstitium. The parenchyma appears to be extensively altered. Tissular MDA gradually increases until it reaches a maximum level (p < 0.01 vs basal) at 48 hours. Positivity for NADPH-d in macrophage and/or fibroblastic cells was evident after 24 hours and increased after 48 hours. CONCLUSIONS: Acute lung injury caused by endotoxin involves both NO and RO degree. Their production is related to different cell types and follows slightly different kinetic.

An antisense oligonucleotide on the mouse Shaker-like potassium channel Kv1.1 gene prevents antinociception induced by morphine and baclofen.

Inactivation of the Kv1.1 gene, which codes for a member of the Shaker-like potassium channels by an antisense oligodeoxyribonucleotide (aODN), was carried out in mice. The effect of this inactivation on analgesia induced by morphine (5-9 mg kg-1 s.c.) and baclofen (2-5 mg kg-1 s.c.) was investigated in the mouse hot-plate test. Mice received a single intracerebroventricular injection of mKv1.1 aODN (0.5, 1.0, 2.0 or 3.0 nmol per injection), degenerated ODN or vehicle on days 1, 4 and 7. A dose-dependent inhibiton of morphine and baclofen antinociception was observed 72 h after the last intracerebroventricular aODN injection, whereas degenerated ODN and vehicle, used as controls, did not affect morphine- and baclofen-induced antinociception. Sensitivity to both analgesic drugs returned to the normal range 7 days after the end of the aODN treatment, which indicated the absence of any irreversible damage or toxicity caused by aODN. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that a decrease in mKv1.1 mRNA levels occurred only in the aODN-treated group, being absent in all control groups. Furthermore, neither aODN, degenerated ODN nor vehicle produced any behavioral impairment of mice. These results indicate that the mKv1.1 potassium channel, whose gene expression we specifically modulated by means of the antisense ODN strategy, plays an important role in central analgesia induced by morphine and baclofen.

Vitamin E protects human skeletal muscle from damage during surgical ischemia-reperfusion.

PURPOSE: The biochemical and morphological alterations induced in lower limb skeletal muscle by ischemia-reperfusion (I-R) during aortic surgery and the effect of vitamin E pretreatment were investigated. METHODS: Two groups of patients undergoing aortic aneurysm resection, one untreated and one treated with vitamin E, were examined. Quadricep muscle biopsies were taken after induction of anesthesia, at the end of ischemia, and after reperfusion. The malondialdehyde (MDA) content and morphology of biopsies were examined to assess peroxidative processes. RESULTS: Ischemia did not induce an increase in MDA content but did increase neutrophil infiltration in muscle fibers of untreated patients. Reperfusion led to a significant increase in MDA content and to intermyofibrillar edema and mitochondrial swelling. The MDA content was not increased during ischemia and neutrophil infiltration was minimal in vitamin E treated patients. At reperfusion, the MDA content, the ultrastructural injuries and neutrophil infiltration were significantly reduced by the treatment. CONCLUSIONS: Vitamin E is effective in reducing the oxidative muscle damage occurring after a period of I-R.

Quantitation of bcl-2 oncogene in cultured lymphoma/leukemia cell lines and in primary leukemia B-cells by a highly sensitive RT-PCR method.

BACKGROUND: The bcl-2 gene, isolated from the t(14;18) chromosomal translocation breakpoint, is able to prevent apoptotic death induced by various stimuli in different tissues. Therefore bcl-2 oncogene expression could be a key parameter for investigating the molecular mechanisms involved in the apoptosis of normal and neoplastic hematopoietic cells. METHODS: In order to evaluate bcl-2 expression in both follicular B-lymphomas carrying or not carrying the 14;18 translocation and in lymphatic leukemias, we optimized an internal standard-based method of reverse transcriptase-polymerase chain reaction (RT-PCR) for the rapid quantitation of bcl-2 mRNA cellular levels. A simple purification of the reverse transcription products resulted in very high PCR efficiency, so that radioactive labelling of the amplification products was avoided. RESULTS: bcl-2 mRNA levels proved to be higher in t(14;18) than in t(14;18) negative cell lines, and higher in primary leukemia pre-B cells than in early-B cells. Tested for sensitivity by identifying minimal residual t(14;18) B cells expressing the bcl-2/IgH gene, this RT-PCR method was able to detect bcl-2/IgH mRNA from just one t(14;18) positive cell out of ten million t(14;18) negative cells. CONCLUSIONS: The RT-PCR method we optimized appears to be suitable for clinical use in both leukemia/lymphoma characterization and in lymphomatous disease follow-up.

Inhibition of MDR1 gene expression by antimessenger oligonucleotides lowers multiple drug resistance.

The multiple drug resistance of neoplastic cells is mediated by overexpression of the human MDR1 gene, which encodes the transmembrane efflux pump P-glycoprotein. In both cell lines and human tumors the MDR phenotype closely correlates with MDR1 mRNA and P-glycoprotein levels. Reversion of the MDR phenotype was attempted in human colorectal adenocarcinoma doxorubicin (Dx)-resistant cells (Lo Vo/Dx) by long-term administration of an equimolecular mixture of three unmodified ODNs (18mer) targeted to adjacent binding sites of the MDR1 mRNA and carried by a synthetic cationic lipid (DOTAP). Three different experimental parameters were used to evaluate the antimessenger agent's effectiveness in comparison with a random sequence ODN: the level of cell resistance to Dx; the level of P-glycoprotein (determined by flow cytometry); the level of MDR1 mRNA (determined by quantitative RT-PCR). Experimental data indicate that the level of both the MDR1 mRNA and the P-glycoprotein is reduced by approximately 50% by treatment of Lo Vo/Dx cells with a 10 microM total concentration of the aODN mixture every 24 h for 15 days. In agreement with these findings, sensitivity to Dx of the antimessenger agent-treated Lo Vo/Dx cells was almost doubled in comparison with random sequence ODN-treated controls.