The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells

PURPOSE: Coronary calcium is a powerful indicator of arteriosclerosis and can be detected very precisely with electron beam tomography. The method can be applied in patients with known coronary artery disease or in asymptomatic patients at risk of arteriosclerotic disease. METHODS: The standard protocol of EBT consists of 30 to 40 slices of 3-mm thickness with a scan time of 100 ms, no overlap. No contrast medium is needed. The total scan can be performed within one breathhold. The calcium score is calculated as described by Agatston. Radiation exposure amounts to 0.8 mSv per total screen. We used spiral CT with and without ECG trigger as an alternative. RESULTS: At the University of Munich we performed an EBT scan of the heart in 1100 patients within the last year. In 567 patients coronary angiography was performed also (+/- 3 days). Confirming previous reports in the literature, we found a correlation of the calcium score with the age and gender of the patients. Severe coronary artery disease (stenoses > or = 75%) was associated with significantly more calcium than less severe CAD. The calcium score did not discriminate between one-, two- and three-vessel disease. The site of calcification does not correlate with the localization of stenoses. Thirty-three percent of the patients with significant coronary artery disease showed a normal age-adjusted calcium score; a total of 8.1% of patients with severe stenoses did not reveal any coronary calcification (score = 0). With asymptomatic patients there are only a few studies available. Soft plaques cannot be detected with EBT, but in most patients soft plaques occur together with hard plaques. Our results show that spiral CT of the newest generation may also be used for calcium screening. There was an excellent correlation of the calcium scores of EBT and spiral CT at all levels of calcification. DISCUSSION: Coronary calcium is a sensitive marker of coronary artery disease. In the clinical setting EBT is indicated in patients with known coronary artery disease (to evaluate prognosis), in patients who are unable to perform a stress test, and in patients with atypical chest pain. However, lack of calcification may be associated with severe stenoses in a minority of patients. The clinical value in asymptomatic patients needs to be defined: randomized studies are necessary. We see a possible indication in patients with known risk factors, in whom primary preventive strategies could be performed more selectively and cost-effectively.     

Taxol induces bcl-2 phosphorylation and death of prostate cancer cells

Treatment of prostate cancer cell lines expressing bcl-2 with taxol induces bcl-2 phosphorylation and programmed cell death, whereas treatment of bcl-2-negative prostate cancer cells with taxol does not induce apoptosis. bcl-2 phosphorylation seems to inhibit its binding to bax since less bax was observed in immunocomplex with bcl-2 in taxol-treated cancer cells. These findings support the use of the anticancer drug taxol for the treatment of bcl-2-positive prostate cancers and other bcl-2-positive malignancies, such as follicular lymphoma.     

Apoptosis: bcl-2 as a key protein for programmed cell death

The aim of the present paper is to present results of sperm morphology using an objective and manual technique by video image. Experiment 1:252 spermatozoa heads were measured in a microscope and in a monitor by each of three independent observers. The results allowed the calibration of an acetate overlay according to the WHO guideline and following the strict criteria. Experiment 2: 10 morphology slides from normal and abnormal patients were studied. These slides were evaluated by three independent observers, each counting at least 200 cells using the calibrate acetate overlay. In the first experiment the calculation of the regression out-put was: constant: 0.24, standard error of Yc: 0.04, R squared: 0.96, X coefficient: 0.36, and standard error of the coefficient: 0.03. In the second experiment, it can be seen that the differences among the operators are not statistically significant and therefore the experiment is independent from the operator. In conclusion, the methodology developed in this paper for the evaluation of morphology would be a good tool for the evaluation of human sperm morphology.     

Polyamine analogue induction of programmed cell death in human lung tumor cells

The naturally occurring polyamines putrescine, spermidine, and spermine are required for cell growth. Based on this requirement, several polyamine analogues that interfere with polyamine function and metabolism have been synthesized as antineoplastic agents. The symmetrically substituted N1,N12-bis(ethyl)spermine (BESpm), and unsymmetrically substituted N1-ethyl-N11-[(cyclopropyl)methyl]-4, 8-diazaundecane (CPENSpm) have previously been shown to cause rapid cytotoxicity of NCI H157 cells, with concurrent high induction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. However, the precise mechanism(s) of the cytotoxic action of the compounds is not known. We now demonstrate that treatment with either BESpm or CPENSpm results in morphological and biochemical changes consistent with the activation of programmed cell death pathways, and that the unsymmetrically substituted CPENSpm more rapidly activates the death program. These studies suggest that the cell type-specific cytotoxicity of these polyamine analogues may be a result of their ability to selectively activate the cell death pathway in sensitive phenotypes and indicate that the relationship between the structure of the polyamine analogues and the ability to induce programmed cell death should be investigated.     

The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat

BACKGROUND: Stimuli that stress cells, including inflammatory cytokines, ultra-violet irradiation, DNA-damaging chemotherapeutic drugs and heat shock, stimulate a recently identified cytoplasmic signaling system that is structurally related to the mitogen-activated protein kinase pathway. This pathway consists of a cascade of protein kinases including stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), and two kinases that activate it, MEKK and SEK/MKK4. Despite rapid progress in delineating the components of this pathway, the cellular consequence of its activation remains to be defined. RESULTS: We have screened cells for defects in SAPK signaling and identified a cell line, previously characterized for its thermotolerance properties, as being more refractive to SAPK activation induced by heat stress than its thermosensitive parental line. Stable expression of dominant inhibiting SEK mutants in thermosensitive parental cells specifically and effectively blocked SAPK activation after heat shock. These lines also became markedly resistant to the cytocidal effects of thermal stress, confirming the phenotype of the thermotolerant line. These cell lines defective in SAPK activation were also resistant to the lethal effects of the DNA-damaging drug cis-platinum. CONCLUSIONS: Experimentally induced stable blockade of SAPK activation in cells with normal thermosensitivity is sufficient to confer resistance to cell death induced by diverse stimuli including heat and the chemotherapeutic agent cis-platinum. These results suggest that activation of the SAPK pathway by diverse cell stressors plays a critical part in mediating the toxicity of these treatments and inducing cell death. SAPK activation in this context could broadly influence cellular response to stress, modulate apoptosis during development or determine the clinical response of tumor cells to cytotoxic therapies.     

Overexpression of a Hu-bcl-2 transgene in Lurcher mutant mice delays Purkinje cell death

Cerebellar Purkinje cells in the heterozygous Lurcher mutant undergo cell autonomous degeneration beginning in the second week of postnatal development and becoming almost total around 30-45 days. The Lurcher mutation was recently identified as gain-of-function defect in the delta 2 glutamate receptor causing a constitutive current leak, suggesting that +/Lc Purkinje cells die by an excitotoxic mechanism. In previous studies we have shown that overexpression of bcl-2, a key regulator of cell death, in the heterozygous Lurcher mutant does not prevent +/Lc Purkinje cell death. To investigate further the mechanisms of +/Lc Purkinje cell death, we have crossed +/Lc mutants with a second line of Hu-bcl-2 transgenics (NSE73a) that shows an earlier onset of transgene expression and higher expression levels. Analysis of eight +/Lc-NSE73a mutants (4 at 2 months and 4 at 5-6 months) showed that Hu-bcl-2 overexpression delayed, but ultimately could not prevent +/Lc Purkinje cell death.     

Neurotoxin-induced cell death in neuronal PC12 cells is mediated by induction of apoptosis

Death of neuronal cells during development and following deprivation of trophic factors is known to occur via an active mechanism requiring RNA and protein synthesis, known as apoptosis. Apoptosis is a form of cell "suicide" whereby the cell decides its own fate by activating a genetic programme of cell death. In contrast, necrosis is a passive uncontrolled form of cell death often observed in response to a toxic insult. Although it is known that neuronal cell death during development occurs by apoptosis, the mechanisms underlying neurotoxin-induced neuronal cell death remain poorly understood. In this study we have examined the mechanism by which 6-hydroxydopamine, a specific neurotoxin for catecholaminergic cells, induces neuronal cell death in vitro. We report that 6-hydroxydopamine induces cell death in the neuronal PC12 cell line via a mechanism which has the characteristic morphological and biochemical hallmarks of apoptosis. PC12 cells induced to die by 6-hydroxydopamine treatment exhibited cell shrinkage, classical chromatin condensation and membrane blebbing. Analysis of DNA integrity from 6-hydroxydopamine-treated cells revealed cleavage of DNA into regular sized fragments, a biochemical hallmark of apoptosis. 6-Hydroxydopamine-induced apoptosis of PC12 cells was suppressed by desipramine, a monoamine uptake inhibitor, suggesting that 6-hydroxydopamine is initiating apoptosis via a specific intracellular mechanism. Aurintricarboxylic acid, a general inhibitor of nucleases, also suppressed 6-hydroxydopamine-induced apoptosis, suggesting the involvement of an endonuclease in the death pathway. The aetiology of idiopathic Parkinson's disease remains uncertain, although evidence suggests that endogenous and/or exogenous toxins may initiate neuronal cell death in this disease. The dopaminergic neurotoxin 6-hydroxydopamine is used to generate animal models of Parkinson's disease in vivo. We have demonstrated that this neurotoxin kills neuronal cells in vitro by an active process of apoptosis. Thus, the possibility exists that cell death in neurodegenerative diseases such as Parkinsonism also occurs in an active manner initiated by as yet unidentified environmental or metabolic toxins. Cell death that involves activation of an apoptotic programme can be modulated by addition of extracellular trophic factors, and is also controlled by the levels of intracellular factors. If neurotoxin-induced apoptosis plays a role in Parkinson's disease the implication is that the neuronal degeneration may be prevented by pharmacological manipulations.     

The role of blood stem cells in hematopoietic cell renewal

It has been the purpose of this keynote address to review available evidence for the notion that the stem and progenitor cells circulating in the peripheral blood play a decisive role in the homeostasis of blood cell formation distributed throughout dozens of bone marrow units in the skeleton. Furthermore, if this notion is correct, one could speculate that the quantity and quality of stem and progenitor cells in the blood should reflect the functional state of the hematopoietic stem cell system throughout the skeletal bone marrow and provide a new tool for the evaluation of alteration in blood cell production. On this basis, the following questions are considered: A) What do we know about the quality and quantity of blood stem cells in steady-state conditions? B) In what way do blood stem cells respond to perturbations of the "steady-state" of blood cell formation? C) Which role do blood stem cells play during hemopoietic development assuming that the establishment of bone marrow hemopoiesis requires the "seeding" of blood stem cells into an appropriate cellular environment? D) What is the role of blood stem cells in hemopoietic regeneration after partial body irradiation with a small volume of marrow (and hence stem cells) protected? and E) What are the mechanisms and/or kinetics of hemopoietic recovery if stem cells introduced into the circulation were collected from exogenous (autologous or allogeneic) sources? In this review presentation, experimental work of our group and of other members of the scientific community is summarized. It becomes obvious that blood stem and progenitor cells play a key role in hematopoietic homeostasis. Furthermore, their physiology and pathophysiology deserve rigorous experimental studies in order to develop a novel tool in the diagnosis and prognosis of neoplastic and non-neoplastic disorders of blood cell formation.     

The action of Bax and bcl-2 on T cell selection

T cell development and selection in the thymus are shaped by the induction of apoptosis. However, a direct role in T cell development and selection for any of the molecules known to regulate apoptosis has remained controversial. We have studied the effect of bax and bcl-2 transgenes in recombination activation gene 1-deficient (RAG-1(-/-)) mice transgenic for the major histocompatibility complex class I-restricted F5 T cell receptor. Overexpression of a bax transgene in the thymus seriously impairs the production of mature T cells, whereas bcl-2 overexpression greatly promotes it. The effect of bax and bcl-2 overexpression on antigen-induced negative selection was studied using fetal thymic organ cultures. This analysis showed that Bcl-2 strongly inhibits negative selection, whereas Bax does not affect it. Our data directly show that Bcl-2 family members have specific roles in T cell selection and also lend support to the hypothesis that Bax and Bcl-2 can antagonize each other's action in a certain apoptosis pathway while in another they can be functionally nonreciprocal.     

Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death

Carbohydrates comprise an extremely important class of biological molecules but little is known about how they mediate their effects. This gap in understanding is largely due to the fact that obtaining pure carbohydrates in amounts large enough for biochemical studies is extremely difficult. The advent of combinatorial strategies to make carbohydrates promises to revolutionize the field of carbohydrate chemistry and biochemistry.     

Apoptotic death of tumor cells correlates with chemosensitivity, independent of p53 or bcl-2

p53 has been implicated as a determinant of chemosensitivity and radiosensitivity. We measured chemosensitivity of human tumor cell lines (n = 11), with or without wild-type p53, following exposure to clinically useful chemotherapeutic drugs (n = 4). Chemosensitivity and apoptosis induction were correlated independently of p53 status or Bcl-2 protein levels in vitro. Wild-type p53 correlated with chemosensitivity in ovarian carcinoma and some Burkitt's lymphoma cells, but not in leukemia or lung cancer. Bcl-2 levels correlated with chemoresistance only in Burkitt's lymphoma. p53-dependent p21(WAF1/CIP1) induction and cell cycle arrest occurred at sublethal doses of chemotherapy, whereas at lethal doses of chemotherapy apoptotic death was observed, consistent with models proposing a relationship between the level of DNA damage versus survival or death. Loss of apoptosis induction was observed in drug-resistant ML-1 and HL-60 leukemia cells, without changes in p53 or Bcl-2. Targeted loss of p53 protein in H460 lung cancer cells using HPV-16 E6 inhibited the etoposide-induced G1 checkpoint but did not decrease chemosensitivity. Our studies suggest that the simple measurement of apoptosis induction may be a useful predictor of chemosensitivity, at least in vitro, and confirm that p53 status and Bcl-2 expression may be useful predictors of chemosensitivity in certain cell types.     

African swine fever virus gene A179L, a viral homologue of bcl-2, protects cells from programmed cell death

The African swine fever virus (ASFV) open reading frame A179L, which is similar to the human proto-oncogene bcl-2, has been cloned and expressed in vaccinia virus under control of the pEIL synthetic early/late promoter. The A179L gene product prevented cell death in HeLa and BSC-40 cells doubly infected with another recombinant vaccinia virus expressing the interferon-induced double-stranded RNA-activated protein kinase (p68 kinase), which activates a rapid cell death characteristic of apoptosis. This finding suggests that the A179L gene has a function similar to that of bcl-2 in preventing apoptosis and may play an important role during productive ASFV infection.     

A common pathway mediates retinoic acid and PMA-dependent programmed cell death (apoptosis) of neuronal cells

To assess the maternal and neonatal risk associated with high-order cesarean sections, a case-control study was carried out in two university affiliated maternity wards. The outcome of 154 pregnancies of women undergoing cesarean section for the 4th time or more was compared with 148 women sectioned for the 2nd or 3rd time and 132 women of similar age and parity after spontaneous birth. The main outcome measures were maternal operative and postoperative morbidity and neonatal prematurity and its complications, Apgar scores, and the need for intensive care. Women undergoing multiple (> or = 4) cesarean sections had significantly more intra-abdominal adhesions (P < 0.0001) than women sectioned for the 2nd or 3rd time. However, the time interval from incision to delivery and the total duration of operation were similar. The postoperative course was not adversely affected by multiple cesarean sections. A high incidence (16.2%) of preterm cesarean deliveries was noted in the study group. This was due to non-elective repeat cesarean delivery rather than to poor timing of scheduled cesarean sections. The significantly increased (P < 0.05) need for neonatal intensive care was explained by the higher occurrence of prematurity. Low Apgar scores (< or = 7) at 1 and 5 min were significantly (P < 0.01) related to multiple cesarean sections, even after controlling for the effect of gestational age. We conclude that multiple cesarean sections pose little risk for the mother, but may be associated with increased neonatal risk, attributed mainly to preterm non-elective cesarean sections.     

Adenovirus-mediated delivery of rhodopsin-promoted bcl-2 results in a delay in photoreceptor cell death in the rd/rd mouse

Gene transfer to retinal cells may provide a means to retard photoreceptor cell death and thus prevent blindness in diseases such as retinitis pigmentosa. We tested the possibility of interfering with apoptotic photoreceptor cell death in the rd mouse through subretinal delivery of a recombinant replication-defective adenovirus containing the human cDNA for bcl-2, Ad.2.5HRPbcl-2. Photoreceptor-specific transgene expression was accomplished through incorporation of the 2.5 kb human rhodopsin upstream fragment (HRP). Ad.2.5HRPbcl-2 was injected alone or in combination with Ad.CMVPDE beta. Ad.CMVPDE beta contains a cDNA encoding the beta subunit of cGMP phosphodiesterase (PDE beta). Recombinant viruses containing lacZ (driven either by the cytomegalovirus (CMV) promoter/enhancer or HRP) and of Ad.CMVPDE beta and vehicle alone were injected in contralateral eyes as control. Injection of Ad.2.5HRPbcl-2 in the rd mouse resulted in histologically detectable rescue lasting 6 weeks after birth. Extent of rescue was not as large as after delivery of wildtype PDE beta, the gene defective in the rd mouse. However, delivery of genes which prevent apoptotic cell death may have broad application to gene therapy of retinal degenerative diseases.     

The short Sendai virus leader region controls induction of programmed cell death

The replication of nonsegmented minus-strand RNA genomes, like that of Sendai paramyxovirus (SeV), are controlled by the short leader regions present at each end of the linear genomes and antigenomes; the left and right promoters (PL and PR), respectively. Wild-type SeV is highly cytopathic in cell culture, because it induces programmed cell death (PCD). We have found that a recombinant SeV (rSeVGP42), in which the first 42 nt of le+ sequences at PL were replaced with the equivalent sequences of PR, and which produces infectious virus in amounts comparable to wild type, does not kill cells. Further, the increasing replacement of the terminal le+ sequences at PL with le- sequences led to a decreasing fraction of infected cells being apoptotic. This property (PCD-), moreover, is dominant in cells co-infected with SeVwt and rSeVGP42, and the mutant virus therefore appears to have gained a function which prevents PCD induced by SeVwt. Even though this virus has not been selected for naturally, it excludes SeVwt during co-infections of cultured cells or embryonated chicken eggs. The noncytopathic nature of cells infected or co-infected with rSeVGP42 leads automatically to stable, persistent infections. The mutation in rSeVGP42 is not in the protein coding regions of the viral genome, but in the 55-nt-long leader region which controls antigenome synthesis from genome templates. The SeV leader regions, which are expressed as short RNAs, thus appear to control the induction of PCD.     

Characterization of the novel focal adhesion kinase RAFTK in hematopoietic cells

Protein tyrosine kinases (PTKs) mediate signals that respond to many pivotal cellular functions. Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and PTKs, is a critical control mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function. The focal adhesion kinase (FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signaling and plays a role in signal transduction pathways mediating cell adhesion, motility and anchorage-independent growth. Recently, we and others have identified a novel protein tyrosine kinase termed RAFTK, (also known as Pyk2 or Cak-beta), which is related to FAK. This review describes the role of RAFTK in various signaling cascades mainly in reference to hematopoietic cell lineages.     

Differential induction of c-Jun NH2-terminal kinase and c-Abl kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin

The c-Abl nonreceptor tyrosine kinase and the c-Jun NH2-terminal kinase (JNK/stress-activated protein kinase) are activated during the injury response to the DNA-damaging agent cisplatin. Loss of DNA mismatch repair activity results in resistance to cisplatin in human cancer cells, suggesting that the mismatch repair proteins function as a detector for cisplatin DNA adducts. To identify signaling pathways activated by this detector, we investigated the effect of the loss of DNA mismatch repair function on the ability of cisplatin to activate the JNK and c-Abl kinases. The results demonstrate that cisplatin activates JNK kinase 3.8 +/- 0.2-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that activation of c-Abl is completely absent in the DNA mismatch repair-deficient cells. Furthermore, the results show that cisplatin-induced activation of JNK occurs through a stress-activated protein kinase/extracellular signal-regulated kinase kinase 1-independent mechanism. We conclude that activation of JNK and c-Abl by cisplatin is in part dependent upon the integrity of DNA mismatch repair function, suggesting that these kinases are part of the signal transduction pathway activated when mismatch repair proteins recognize cisplatin adducts in DNA.