Apoptosis and its role in human disease

In a landmark paper published over two decades ago, Kerr et al. proposed the term apoptosis "for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations". In the ensuing years, this natural cell death process was studied at the basic science level, primarily with a view to understanding its roles in cancer and in the development and maintenance of the immune system. More recently, however, evidence has suggested a role for the failure of normal apoptosis control in many of the major diseases of the industrialized world. Though complex, apoptosis appears amenable to therapeutic intervention. The range of modern pharmaceutical strategies available to treat such disregulated gene-directed processes offers promise for advances in the control of cancer, immune system and neurodegenerative disorders, heart disease, and perhaps even the aging process itself.     

Galectin-1 and -3 in cells of the first trimester placental bed

The beta-galactoside-binding proteins galectin-1 and -3 are thought to modulate cell-extracellular matrix interactions in cell adhesion and migration. In this study, their occurrence in human trophoblast has been investigated. In the first trimester placenta galectin-1 is expressed in the cytotrophoblast of the mid and distal cell columns, but absent from the villous and proximal column cytotrophoblast. The villous syncytiotrophoblast was also positive. Galectin-3, on the other hand, was uniformly localized in the villous cytotrophoblast and mid and distal cell columns. Immunolocalization of these proteins in placental bed tissue has shown that galectin-1 and -3 are not present in cytokeratin-positive interstitially migrating cytotrophoblast. The co-localization of galectin-1 with extracellular laminin in cultures of cytotrophoblast, choriocarcinoma or decidual stromal cells is consistent with a role in the organization of extracellular matrix and the regulation of cell motility.     

Cocaine-sensitive sigma-receptor and its interaction with steroid hormones in the human placental syncytiotrophoblast and in choriocarcinoma cells

The expression and ligand binding characteristics of sigma-receptors in human placental syncytiotrophoblast and choriocarcinoma cells were investigated using haloperidol as a ligand. Haloperidol bound to purified placental brush border membranes with high affinity; the apparent dissociation constant for the process was about 3 nM. These binding sites were not related to dopamine (D2) and serotonin (5-HT2) receptors nor to serotonin and norepinephrine transporters. The ligands of sigma-receptors [3.g. (+)-3-(3-hydroxyphenyl)N-(1-propyl)piperidine, 1,3-di-(2-tolyl)guanidine, clorgyline, rimcazole, and dexromethorphan] were very potent in competing with haloperidol for the binding sites. The binding sites were detected not only in the brush border membrane, but also in intracellular membranes. The rank order of potency of various sigma-receptor ligands to inhibit haloperidol binding indicated that placental sigma-receptors belong to the sigma 1 subtype. Cocaine and its analog RTI-55 [2 beta-carbomethoxy-3 beta-(4-iodophenyl-) tropane] inhibited the binding of haloperidol to placental membranes with appreciable potency. The steroid hormones, progesterone and testosterone, were also potent inhibitors, and the inhibition constant for progesterone was 0.3 microM, a concentration much smaller than that found in plasma during pregnancy. The inhibition was competitive. beta-Estradiol and a number of other steroids were relatively much weaker inhibitors than progesterone and testosterone. Phenytoin and neuropeptide-Y did not interact with sigma-receptors in placenta. The choriocarcinoma cell line JAR was also found to express sigma-receptors in the plasma membrane as well as in intracellular membranes. The characteristics of the receptors in this cell were qualitatively similar to those of the receptors in normal placenta, including subtype identity and interaction with cocaine and progesterone. Interestingly, however, all sigma-receptor ligands interacted with the receptors in the JAR cell with much higher affinity than with the receptors in normal placenta. It is concluded that the placental syncytiotrophoblast and choriocarcinoma cells express cocaine-sensitive sigma-receptors and that progesterone is most likely an endogenous ligand for these receptors.     

Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed.     

Cytophotometric determination of DNA concentration in the nuclei of the human placental trophoblast in late toxemias of pregnancy

The distribution of trophoblast nuclei of the human placenta by their ploidy was studied in normal pregnancy and in late pregnancy toxemias by the monowave emthod of cytophotometry in visible light. In the plasmodiotrophoblast a pronounced increase in the amount of nuclei in the S-phase (up to 40%) was noted in late toxemias which suggests a considerable activation of the DNA synthesis. The placenta cytotrophoblast in late toxemias is substantially different from that in normality. The Langhans cells disappear almost completely while in normality they are present in young terminal villi till the very end of pregnancy. The amount of the island cytotrophoblasts is much greater, the distribution of nuclei according t the DNA content being of absolutely different character than in normality. A great amount of hypodiploid nuclei (up to 36%) was observed which are absent in normal conditions as well as much greater fluctuations in the amount of diploid nuclei (from 4% to 37%) as compared with the normal amount (25.9%). These data show a high amitotic activity in the island cytotrophoblast in late toxemias. The above changes in the plasmodiotrophoblast and cytotrophoblast might be considered as a compensatory reaction of the placenta to a considerably increased vacuolization and deep atrophic processes in degenerating villi.     

Hyaluronan, CD44 and its variant exons in human trophoblast invasion and placental angiogenesis

Both hyaluronan and one of its receptors, CD44, can be demonstrated in the early human conceptus and in placental stroma. The variants of CD44 resulting from variable exon splicing are found in metastasizing human malignancies and are also involved in hyaluronan uptake and degradation. The resulting hyaluronan fragments are known to be highly angiogenic. We postulated that the self-limited process of trophoblast invasion of the uterine decidua results in part from the strategy of alternative splicing of CD44, similar to that used by invasive cancer cells in the course of metastatic spread and possibly angiogenesis. Monoclonal antibodies specific for CD44s and for an exon expressed during metastatic tumour progression, CD44v7, were used to examine this hypothesis. In this study we found human trophoblasts, for the first time, to express CD44. Intermediate trophoblasts of first and second trimester exhibited the standard form of CD44 while extravillous trophoblasts, which are responsible for the invading characteristics of the placenta, were positive for the alternatively spliced form, the CD44v7-8. Moreover, in the case of placenta accreta there was a prominent membrane staining of the trophoblasts that were embedded in the fibrin layer over the myometrium. The highly metastatic choriocarcinoma cells also expressed CD44v7-8. We propose, therefore, that the invading trophoblasts utilize the alternatively splicing machinery. These cells retain their invasive capabilities through the permissive ECM by carrying the CD44v7-8 isoform, which binds weakly to hyaluronan and thus prevents it from being degraded by intracellular hyaluronidase.     

Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in vitro and models the placental defects that occur in preeclampsia

During normal human pregnancy a subpopulation of fetal cytotrophoblast stem cells differentiate and invade the uterus and its arterioles. In the pregnancy disease preeclampsia, cytotrophoblast differentiation is abnormal and invasion is shallow. Thus, the placenta is relatively hypoxic. We investigated whether lowering oxygen tension affects cytotrophoblast differentiation and invasion. Previously we showed that when early gestation cytotrophoblast stem cells are cultured under standard conditions (20% O2) they differentiate/invade, replicating many aspects of the in vivo process. Specifically, the cells proliferate at a low rate and rapidly invade extracellular matrix (ECM) substrates, a phenomenon that requires switching their repertoire of integrin cell-ECM receptors, which are stage-specific antigens that mark specific transitions in the differentiation process. In this study we found that lowering oxygen tension to 2% did not change many of the cells' basic processes. However, there was a marked increase in their incorporation of [3H]thymidine and 5-bromo-2'-deoxyuridine (BrdU). Moreover, they failed to invade ECM substrates, due at least in part to their inability to completely switch their integrin repertoire. These changes mimic many of the alterations in cytotrophoblast differentiation/invasion that occur in preeclampsia, suggesting that oxygen tension plays an important role in regulating these processes in vivo.     

Apoptosis in the liver and its role in hepatocarcinogenesis

Apoptosis seems to be the predominant type of active cell death in the liver (type I), while in other tissues cells may die via biochemically and morphologically different pathways (type II, type III). Active cell death is under the control of growth factors and death signals. In the liver, endogenous factors, such as transforming growth factor beta 1 (TGF-beta 1), activin A, CD95 ligand, and tumor necrosis factor (TNF) may be involved in induction of apoptosis. Release and action of these death factors seems to be triggered by exogenous signals such as withdrawal of hepato-mitogens, food restriction, etc. During stages of hepatocarcinogenesis, not only DNA synthesis but also apoptosis gradually increase from normal to preneoplastic to adenoma and carcinoma tissue. Also, in human carcinomas, birth and death rates of cells are several times higher than in surrounding liver. (Pre)neoplastic liver cells are more susceptible than normal hepatocytes to stimulation of cell replication and of cell death. Consequently, tumor promoters may act as survival factors, i.e., inhibit apoptosis preferentially in preneoplastic and even in malignant liver cells, thereby stimulating selective growth of (pre)neoplastic lesions. On the other hand, regimens favoring apoptosis and lowering cell replication may result in selective elimination of (pre)neoplastic cell clones from the liver. Finally, we have studied the first stage of carcinogenesis, namely the appearance of putatively initiated cells after a single dose of the genotoxic carcinogen N-nitrosomorpholine (NNM). Most of these cells were found to be eliminated by apoptosis, suggesting that initiation, at the organ level, can be reversed at least partially by preferential elimination of initiated cells. These events may be regulated by autocrine or paracrine actions of survival factors.     

Isolation of DNA polymerase beta from human placenta and study of its substrate specificity

A method of purification of DNA polymerase beta with a specific activity of 1300 units/mg from human placenta was developed. The enzyme preparations do not contain any other DNA polymerase activities and any nuclease contaminations degrading nucleic acids. On the basis of analysis of several standard parameters we conclude that the purified enzyme is polymerase beta. The optimal conditions of polymerization were established, and a comparison of the relative rates of polymerization with various template-primer complexes was carried out. Activated DNA was shown to be the optimal substrate in the presence of MgCl2, and poly(dA).oligo(dT) in the presence of MnCl2. The activation energies of polymerization for different template-primers were estimated.     

Apoptosis, necrosis, and proliferation: possible implications in the etiology of keloids

Keloids are collagenous lesions acquired as a result of abnormal wound heating. In this study we have assessed the potential role of proliferation, apoptosis, and necrosis in keloids. Samples were immunolabeled for proliferating cell nuclear antigen or DNA strand breaks or stained with acridine orange. Proliferating cells were observed in the basal layer of the epidermis and fibroblasts in the dermis, the numbers of the latter being increased in comparison with normal skin. No proliferating cells were observed in the central region of the keloid. In normal skin, apoptotic cells were restricted to the basal layer of the epidermis. In keloid samples, numerous apoptotic cells were observed in the epidermis and dermis; the number and distribution of positive cells decreased more distal to the keloid lesion. Apoptotic endothelial cells of a small proportion of blood vessels in the dermis were also observed. Evidence of necrosis was also seen in the dermis. These results suggest that, with maturity, progressive cell degeneration primarily by apoptosis results in clearance of certain cellular populations resulting in the typical keloid lesion. However, the persistence of fibroblast proliferation at the dermal/keloid interface propagates the fibrosis.     

Association of circulating endothelium and noradrenaline with increased calcium-channel binding sites in the placental bed in pre-eclampsia

PURPOSE: During presurgical evaluation, 14 patients with medically intractable focal epilepsies underwent magnetoencephalographic (MEG) recordings to localize the epileptogenic focus. To increase the number of epileptiform discharges required for MEG analysis, methohexital a short-acting barbiturate that is known to activate epileptiform activity, was used. Additionally, we investigated the spike-provoking properties of clonidine in comparison to methohexital. METHODS: After oral premedication with clonidine, short-lasting anesthesia was provided by intravenously administered methohexital. The number and location of epileptiform MEG discharges were assessed after clonidine premedication and during methohexital anesthesia. Results were compared with baseline MEG recordings. RESULTS: Methohexital increased the frequency of focal epileptiform discharges in eight of 13 patients (one of the 14 patients did not receive methohexital after premedication with clonidine). Additionally, premedication with clonidine was found to increase focal epileptiform discharges in nine of 14 patients. When compared with baseline MEG recordings, recordings after treatment with both clonidine premedication and methohexital anesthesia showed a significant increase in the total number of epileptiform signals and the number of spikes contributing to MEG source localizations. CONCLUSIONS: This study confirms the selective proconvulsant effects of methohexital on the epileptogenic focus as suggested previously by EEG and electrocorticogram (ECoG) investigations. Additionally, our data establish for the first time that clonidine increases epileptiform activity in patients with seizure disorders. These results indicate that clonidine is suited as an activating agent for the localization of epileptogenic foci by means of MEG. This effect of clonidine on specific epileptic activity also indicates that clonidine should be used with caution as an antihypertensive drug in patients with seizure disorders.     

Apoptosis in cultured human colon cancer cells induced by combined treatments with 5-fluorouracil, tumor necrosis factor-alpha and interferon-alpha

An experimental study of Allergan's Oxysept Comfort system was performed by measuring the slight reddish hue that appears in the disinfecting solution, indicating to the users that their lenses are again ready to be worn. The temporal evolution of the color of the solution has been measured under standardized conditions and analyzed in the CIELAB system, from the perspective of the typical threshold discrimination of the human eye. Color differences between neutralized and non-neutralized solutions occurred in an appropriate direction of the color space to enhance discrimination and were clearly perceptible by normal observers (greater than 9.7 +/- 1.2 CIELAB units). Colorimetric analyses have been used to draw conclusions regarding observers with defective color vision. The color of the solution changes abruptly, approximately 25 min after the neutralization process begins, and remains nearly constant after about 60 min, this agreeing well with the temporal evolution of the hydrogen peroxide concentration.     

The lecithin/spingomyelin quotient as determined using a modified method and human placental lactogen in the amniotic fluid

Thirty amniotic fluid samples in late gestational age were analysed for HPL-values and the L/S-ratio. The pregnant women used in this study were healthy except three patients who developed a rh-incompatibility. The amniotic fluid was only obtained by abdominal amniocentesis. We found a decreasing tendency of the HPL-values in late pregnancy. The L/S-ratio increased. A significant correlation of both values could not be observed. In all three cases of rh-incompatibility the decrease of HPL and the increase of the L/S-ratio occurred obviously earlier and steeper. Further studies will prove the significance of these results.     

Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopathogenicity

The hallmark of Legionnaires' disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellular L. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophila correlates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.     

Apoptosis induced by sulindac sulfide in epithelial and mesenchymal cells from human abdominal neoplasms

A tadpole of bullfrog, Rana catesbeiana, is originally covered with the larval skin over its entire body. Drastic changes arise in both the epidermis and the subcutaneous connective tissue at an early developmental stage, producing the precursor of adult type skin (pre-adult skin). It was found that calcium is a useful probe to detect the region where the precursor formation has occurred because its deposition in the upper part of subcutaneous collagen bundles coincides with the appearance of the pre-adult skin. Whole-mount in situ staining of tadpoles with alizarin red S revealed the initiation site of the premetamorphic transformation of the larval skin into the adult precursor and its ensuing region-dependent expansion. The pre-adult skin first emerged at TK II to III (TK, Taylor and Kollros staging) t lateral sides of the body, which led us to postulate that 'the center for premetamorphic skin transformation' is formed at the specific site in this region. This center moved dorsally and then ventrally, then reached to the most proximal region of the tail, yielding a unique sequential conversion pattern by around TK V when the conversion was completed in the trunk. The present study also visualized the process of the hindlimb skin transformation.     

Molecular anatomy of the perivascular sheath in human placental stem villi: the contractile apparatus and its association to the extracellular matrix

In previous studies, we have shown that smooth muscle cells and myofibroblast subpopulations of the perivascular stem villous sheath of the human placenta contain focal adhesion plaques and talin immunoreactivity. The close association of these cells to elastic and collagen fibres have led to the assumption of a functional myofibroelastic unit within the perivascular stem villous sheath. Interactions between the extracellular matrix and smooth muscle cells depend on a variety of structural protein assemblies. In the present study, we examined, by immunocytochemistry, whether the molecular assembly of extracellular matrix proteins and molecules of focal adhesions, known to be essential for signal transduction in smooth muscle cells, are also found in smooth muscle cells of the perivascular stem villous sheath of the human placenta. Vascular and extravascular smooth muscle cells were immunoreactive for alpha-actinin, vinculin, paxillin and tensin, the integrin chains alpha1 and beta1, and the basement membrane components laminin and heparan/-chondroitin sulfate proteoglycan perlecan. pp125(FAK) did not react. In the extracellular matrix of blood vessel walls and the perivascular stem villous sheath, we found immunoreactivity of fibronectin and collagen types I, VI and undulin (collagen type XIV). From our data we conclude that within the perivascular stem villous sheath, there exists a system of signal transduction molecules, indicating a cross talk between the smooth muscle cells of this sheath and their surrounding extracellular matrix.     

Arsenic toxicity is enzyme specific and its affects on ligation are not caused by the direct inhibition of DNA repair enzymes

There is an increasing awareness that the exclusion of women from clinical trials may lead to inaccurate application of drug therapy in women. Gender and estrus cycle differences in the pharmacokinetics and pharmacodynamics of drugs in animals have been appreciated for over 60 years, but investigation into these differences in humans has only recently occurred. It is postulated that hormonal fluctuations within the menstrual cycle phase may be a primary cause of documented gender differences in the pharmacokinetics and pharmacodynamics of drugs. Existing data suggest that menstrual cycle variations do occur in renal, cardiovascular, haematological and immune systems. These physiological changes could potentially impact on the pharmacokinetics or pharmacodynamics of drugs by altering properties, such as protein binding or the volume of distribution, and thereby causing significant effects at various times during the menstrual cycle. However, systematic investigations of physiological variability throughout the menstrual cycle are limited. Fluctuations in symptom severity and clinical course coinciding with the menstrual cycle phase have been seen in some diseases. Hormonal fluctuations within the menstrual cycle have been postulated to cause disease exacerbation. They may also worsen disease severity by impacting on the pharmacokinetics or pharmacodynamics of the medication. Menstrual cycle hormonal changes may influence drug absorption, distribution, metabolism or excretion. In vivo data to demonstrate an effect of endogenous estrogen or progesterone on pharmacokinetics are limited and contradictory. Systematic investigations of specific pharmacokinetic and pharmacodynamic changes within the menstrual cycle are lacking. Most published studies have been conducted with small numbers of women and a limited numbers of menstrual cycle phases within 1 menstrual cycle. These design problems have resulted in incomplete data for assessing the effects of the menstrual cycle. To date, there are no demonstrated clinically significant changes that occur in the absorption, distribution or elimination of drugs. With respect to drug metabolism, data are exceedingly sparse and have been collected in a suboptimal fashion. Standardisation of study design and analyses in systematic investigations of the influence of the menstrual cycle on drug pharmacokinetics and pharmacodynamics are needed.