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Setting: A tertiary care and research institution in Italy., Background: Small DNA fragments from cells dying throughout the body have been detected in urine (transrenal DNA [Tr-DNA])., Objective: To test the hypothesis that Mycobacterium tuberculosis Tr-DNA could be detected in the urine of pulmonary tuberculosis (TB) patients., Design: We studied 43 patients with culture-confirmed pulmonary TB with no evidence of extra-pulmonary involvement, 10 patients with pulmonary diseases other than TB and 13 healthy controls. DNA was extracted from urine and analysed by semi-nested polymerase chain reaction (PCR)., Results: M. tuberculosis-specific sequences were found in the urine of 34 of 43 (79%) TB patients studied, whereas all controls were negative. The transrenal nature of M. tuberculosis DNA was demonstrated by two lines of evidence: first, separate analysis of supernatants and sediments from eight of the study patients found seven positive supernatants but only two matched positive sediments. Second, M. tuberculosis-specific sequences were amplified by semi-nested PCR with primers designed for short but not large amplicons., Conclusion: Small M. tuberculosis DNA fragments may be detected in the urine of a significant proportion of patients with pulmonary TB. If these observations are confirmed by larger studies, Tr-DNA technology could represent a new approach for detecting pulmonary M. tuberculosis infection.

Transrenal DNA (Tr-DNA) is a recently discovered class of extracellular urinary DNA that originates from cells dying throughout the body. Postapoptotic DNA is known to appear in the circulating plasma, but it is now recognized that a portion of these fragments cross the kidney barrier and appear in urine in the form of 150-200-bp fragments. Tr-DNA containing fetal sequences has been isolated from the urine of pregnant women, tumor-specific mutations have been detected in Tr-DNA from patients with colon and pancreatic tumors, and donor DNA has been found in Tr-DNA isolated from recipient urine. Furthermore, proviral HIV DNA, bacterial and parasite DNA sequences have been detected in Tr-DNA from infected patients. Potential applications of Tr-DNA-based tests cover a very broad area of molecular diagnostics and genetic testing, including prenatal detection of inherited diseases, tumor diagnostics and therapeutic monitoring and detection of infectious agents. The Tr-DNA test is expected to have utility in treatment monitoring, transplantation monitoring, drug development and broad public health screening, where a noninvasive, common-platform diagnostic technology has particular value. This review describes some of the highlights of Tr-DNA technology applications, advantages over existing technologies and potential problems anticipated in test development.

DNA fragments from cells that have died throughout the body not only appear in the bloodstream but also cross the kidney barrier into the urine. The relatively low molecular weight (150-200 bp) of this Transrenal DNA should be considered when deciding on methods of isolation and analysis. In particular, if polymerase chain reaction (PCR) is used for amplification and detection of specific sequences, then the reduction of amplicon size will significantly enhance sensitivity. Detection of DNA mutations is also made more difficult by the presence of a large excess of a wild-type allele. Using K-RAS mutations as an example, two ways around this problem--enriched PCR and stencil-aided mutation analysis-are described, based on selective pre-PCR elimination of wild-type sequences.

A small portion of DNA from apoptotic cells escapes complete degradation, appears in blood as oligonucleosomal-size fragments, is excreted in the urine, and can be used for diagnostic purposes. More detailed study revealed that transrenal DNA (Tr-DNA) belongs to a relatively low molecular-weight (150-250 bp) fraction, thereby requiring more careful attention to methods employed for purification and analysis. For example, here it is demonstrated that the QIAamp blood kit purifies primarily high molecular-weight DNA from serum, whereas the Guanidine/Promega Wizard Resin (GITC/WR) method purifies primarily low molecular-weight DNA. As a result, sensitivity in detection of K-RAS mutations in serum of patients with colorectal tumors is significantly higher with DNA isolated with the GITC/WR method than with the QIAamp kit. Amplicon size is also extremely important in analysis of Tr-DNA, because the shorter the amplicon, the higher is the sensitivity of biomarker detection in Tr-DNA. One hundred fifty-seven and 87 bp amplicons were employed for detection of mutant K-RAS in DNA isolated from 0.1 mL of urine obtained from 15 patients with pancreatic cancer. Mutant K-RAS was found in Tr-DNA of 3 and 10 patients with the long and short amplicons, respectively. The sensitivity and specificity of detection of mutant sequences are reduced in the presence of high excess of a respective wild-type allele, but they can be significantly increased through application of enriched polymerase chain reaction (PCR), peptide nucleic acid (PNA) clamped PCR, and/or stencil-aided mutation analysis (SAMA), based on selective pre-PCR elimination of wild-type sequences.

Apoptosis continues to be a controversial concept and subject of debate among scientists regarding its value as the basis for new therapeutic strategies. Today, it is widely accepted that the death of cardiac myocytes under a variety of conditions appears to be apoptotic based on a variety of criteria. However, the significance of these observations and how the insights into apoptotic molecular pathways may provide novel therapeutic targets remains to be determined. It is important to reconsider the pertinent underlying mechanisms of apoptosis regulation, and how these molecular pathways may be viewed in the functioning, intact heart. This knowledge can be applied in pursuit of practical goals in a search for new ways to prevent myocardial damage following such injuries as ischaemia/reperfusion or exposure to cardiotoxic drugs. Although recent literature contains reports of positive findings, there has not yet been a rigorous application of the model of apoptosis in the myocardium, and the potential for development of new therapeutic strategies is not yet understood.

Cardiomyopathies are observed with increasing frequency in association with AIDS and HIV infection. Although indirect evidence exists suggesting an association between apoptosis regulation and HIV infection, there is yet no direct evidence that HIV-associated cardiomyopathies involve increased level of apoptosis in the heart. However, since it is now known that apoptosis plays a significant role in heart injury associated with other conditions such as ischemia/reperfusion and heart failure, there is a possibility that dysregulation of apoptosis plays a similarly important role in HIV-associate cardiomyopathies. Here we will briefly review the evidence that apoptotic death of cardiomyocytes occurs and what novel therapeutic strategies may be suggested.

It is well documented that plasma contains DNA from tissues throughout the body, including developing fetuses, and tumors. A portion of this DNA crosses the kidney barrier and appears in urine (i.e., transrenal DNA). However, molecular, cellular, and physiological mechanisms of the circulating DNA phenomenon and renal clearance are in an early phase of investigation. Here, we discuss possible forms of circulating DNA, factors affecting representation of different tissues and genomic sequences in plasma DNA, possible mechanisms of renal DNA clearance, and technical problems encountered in DNA isolation from urine. We suggest that apoptotic cells are an important source of DNA in both plasma and urine. Further analysis of the data has led us to propose that a significant portion of circulating DNA can be represented in apoptotic bodies.

Peroxysomicine A1, a novel potential anticancer compound induced cell death in established cell lines and in a primary culture of rat neonatal cardiomyocytes. Non-transformed cells are less sensitive to the compound than transformed cell lines. Fluorescent microscopy of dying cells stained with DNA-specific dyes revealed chromatin condensation and nuclear fragmentation as well as membrane blebbing characteristic of apoptosis. Flow cytometry of cells treated with peroxysomicine A1, demonstrated appearance of cells containing less than 2C DNA, that indicated degradation of nuclear DNA, another hallmark of apoptotic cell death. Z-VAD, a nonspecific caspase inhibitor, prevented DNA fragmentation but not cell death registered by permeabilization of cell outer membrane. Peroxysomicine A1 also inhibited proliferation of various cell lines. Flow cytometry analysis showed significant accumulation of dividing cells in G2/M phases of cell cycle indicating, most likely delay in G2. These results provide initial insight into the mechanisms of action of peroxysomicine A1 and suggest that peroxysomicine A1 is a potent inhibitor of cell proliferation and inducer of apoptosis and may be a useful antineoplastic chemotherapeutic agent.

The ability of a previously described soy-derived antiapoptotic fraction (SDAAF), a soy water extract (Lexirin), and raw soy flour to inhibit methotrexate (MTX)-induced gastrointestinal damage was evaluated by histological examination of duodenal/jejunal sections from MTX-treated rats. Male Sprague-Dawley rats were fed diets containing casein as a sole protein source or diets supplemented with fractions isolated from soy (SDAAF or Lexirin) before and after MTX treatment. The soy fractions were also shown to inhibit serum deprivation-induced programmed cell death (apoptosis) in mouse embryonic C3H10T1/2 cells. Protein sequence (Lexirin) and enzyme activity (Lexirin and SDAAF) were also analyzed. Rats that received SDAAF- and Lexirin-supplemented diets had significantly reduced necrotic and apoptotic damage in the duodenal mucosa, as demonstrated by difference in villi height, mitotic activity, epithelial cell height, and inflammatory response.

Previous studies have shown that extracts from soy possess potent antiapoptotic activity in in vitro and in vivo models. We recently reported that this antiapoptotic activity can be attributed to the presence of specific phospholipids. In this study, a conventional preparation of the soy-derived Bowman-Birk inhibitor (BBI) was tested for antiapoptotic activity in a C3H/10T1/2 cell serum deprivation assay. The BBI preparation was separated into lipid- or protein-containing fractions by organic extraction. The lipid fraction contained only antiapoptotic activity; the protein fraction contained only enzyme inhibition activity. We therefore conclude that the antiapoptotic activity of the BBI preparation is due to specific phospholipids that copurify with BBI. These phospholipids retain their antiapoptotic activity after autoclave treatment, whereas autoclave treatment of the protein fraction results in a loss of its enzyme inhibition activity.

The phenomenon of aging is distinct from processes associated with advanced age known to increase risk of diseases, such as cancer. Furthermore, the process of aging is not necessarily related to phenomena such as in vitro replicative senescence; however, any unifying hypothesis of aging must account for all age-dependent phenomena, including senescence. It is proposed that apoptosis forms the ultimate protective process for preservation of phenotypic fidelity in multicellular organisms since it is the process by which the organism detects damage and replaces the defective cell. Time-dependent degeneration of apoptosis control is the rate-limiting step in the process of aging.

We have functionally expressed the human cDNA encoding the putative lysophosphatidic acid (LPA) receptor Edg-2 (Vzg-1) in Saccharomyces cerevisiae in an attempt to determine the agonist specificity of this G-protein-coupled receptor. LPA activated the pheromone response pathway in S. cerevisiae expressing Edg-2 in a time- and dose-dependent manner as determined by induction of a pheromone-responsive FUS1::lacZ reporter gene. LPA-mediated activation of the pheromone response pathway was dependent on mutational inactivation of the SST2 gene, the GTPase-activating protein for the yeast G alpha protein (the GPA1 gene product). This indicates that, in sst2 delta yeast cells, Edg-2 can efficiently couple to the yeast heterotrimeric G-protein in response to LPA and activate the yeast mitogen-activated protein kinase pathway. The Edg-2 receptor showed a high degree of specificity for LPA; other lyso-glycerophospholipids, sphingosine 1-phosphate, and diacyl-glycerophospholipids did not activate FUS1::lacZ. LPA analogs including a cyclic phosphoester form and ether-linked forms of LPA activated FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ, suggesting a role for the side chain in ligand binding or receptor activation. These results indicate that Edg-2 encodes a highly specific LPA receptor.

Quiescent mouse embryonic C3H/10T1/2 cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T1/2 cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T1/2 cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T1/2 cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of beta-catenin, suggesting that SARPs interfere with the Wnt-frizzled proteins signaling pathway.

Involvement of ceramide signaling in the initiation of apoptosis induction in myocardial cells by in vitro and in vivo ischemia and reperfusion was analyzed. Synthetic cell permeable C2-ceramide induced apoptotic death of rat neonatal cardiomyocytes in vitro. In vitro ischemia (oxygen/serum/glucose deprivation) led to a progressive accumulation of ceramide in cardiomyocytes. After 16 hours of simulated in vitro reperfusion (readdition of oxygen, serum and glucose), the level of ceramide in surviving cells was found to have returned to baseline, whereas, levels in nonadherent dead cells remained high. In the rat heart left coronary artery occlusion model, ischemia with the subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells as demonstrated by DNA electrophoresis and measurement of soluble chromatin degradation products. The content of ceramide in ischemic area was elevated to 155% baseline levels at 30 minutes, and to 330% after 210 minutes of ischemia. Ischemia (30 minutes) followed by reperfusion (180 minutes) increased the ceramide level to 250% in the ischemic area. The combination of results obtained in both in vitro and animal models demonstrate for the first time that ceramide signaling can be involved in ischemia/reperfusion death of myocardial cells.

Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its anti-apoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.

Interferon (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p53 mutants, to tumor necrosis factor-alpha-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p53 function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP32 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-3 (ICE-LAP3, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p53. The IFN-responsive transcriptional activator interferon regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p53-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.

Background: Apoptosis (programmed cell death) occurs as a consequence of global organ ischemia during isolation and storage prior to transplantation. If apoptosis is inhibited during ischemia, organ preservation should be improved, and the length of time for permissible storage may be increased. The objective of this study was to test the effect of a newly developed antiapoptotic compound, LXR-015, during extended hypothermic liver preservation., Methods: Three groups of 12 rats each were studied. In the normal group, liver function was studied immediately after harvesting. In the study group, harvested livers were flushed with Euro-Collins solution (30 ml/kg body weight) containing LXR-015 at a concentration equivalent to 9 mg/kg animal body weight (300 microg/ml). The livers were then stored at 4 degrees C for 24 hr before liver function was studied. In the control group, harvested livers were flushed with Euro-Collins solution without LXR-015 and then stored at 4 degrees C for 24 hr before liver function was studied., Results: Portal venous flow was higher (P<0.05) in the normal and study groups compared with the control group. Portal venous resistance was lower (P<0.05) in the normal and study groups compared with the control group. Liver tissue oxygen consumption in the study group was significantly higher than in both the normal and control groups (P<0.05). Liver enzyme production (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase) was higher in the control group than in either the study or normal group (P<0.05). Bile production in both the normal and study groups was higher than in the control group (P<0.05). The liver tissue wet to dry weight ratio in both the normal and study groups was lower than in the control group (P<0.05). Histopathology studies revealed fewer apoptotic bodies (P<0.05) in both the normal (1.70+/-0.15 per high-power field) and study groups (2.08+/-0.10 per high-power field) than in the control group (7.92+/-.33 per high-power field)., Conclusions: Adding an antiapoptotic compound, LXR-015, to Euro-Collins solution significantly improves hypothermic preservation of the rat liver compared with Euro-Collins solution alone.

The ability of a soy-derived antiapoptotic fraction to inhibit methotrexate-induced gastrointestinal toxicity was examined. Male Sprague-Dawley rats treated with methotrexate were fed diets containing casein as a sole protein source or diets supplemented with a protein-phospholipid fraction isolated from soy flour. This soy fraction has also been shown to inhibit serum deprivation-induced programmed cell death (apoptosis) in the mouse embryonic C3H10T1/2 cell. Rats that received high doses of the soy-derived antiapoptotic fraction-supplemented diets experienced significantly less weight loss and diarrhea and better maintained their pretreatment appetite.

Lysophosphatidic acid (1-acyl-2-lyso-snglycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl- currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5-11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl- current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.

Despite the clinical importance of cardiomyocyte death following ischemia and reperfusion, little is known about the nature of the process. In primary rat neonatal cardiomyocyte cultures, cell death was induced by ischemia (deprivation of oxygen, serum and glucose) and reperfusion. We report here that ischemia induced primarily necrosis, whereas subsequent reperfusion induced apoptosis. Apoptosis of rat neonatal cardiomyocytes could not be prevented by protein synthesis inhibitors, suggesting that molecular components of the apoptotic pathway pre-exist in these cells. IGFs and calpain inhibitors had no effect on necrotic death during ischemia, but they significantly reduced apoptotic death during reperfusion. These results support the concept that inhibition of post-ischemic apoptotic death in the myocardium may provide a valuable new therapeutic strategy for the treatment of acute myocardial ischemia.

Members of the Bcl-2 family of proteins are characterized by their ability to modulate cell death. Bcl-2 and some of its homologues inhibit apoptosis, whereas other family members, such as Bax, will accelerate apoptosis under certain conditions. Here we describe the identification and characterization of a complementary DNA that encodes a previously unknown Bcl-2 homologue designated Bak. Like Bax, the bak gene product primarily enhances apoptotic cell death following an appropriate stimulus. Unlike Bax, however, Bak can inhibit cell death in an Epstein-Barr-virus-transformed cell line. The widespread tissue distribution of Bak messenger RNA, including those containing long-lived, terminally differentiated cell types, suggests that cell-death-inducing activity is broadly distributed, and that tissue-specific modulation of apoptosis is controlled primarily by regulation of molecules that inhibit apoptosis.

Apoptosis in embryonic C3H/10T1/2 (clone 8) cells is marked by specific changes in morphology and DNA fragmentation that differ from those found in apoptotic thymocytes. These results demonstrate that ultrastructural changes within the nucleus associated with endonucleolytic degradation are linked with structural degradation at higher levels of chromatin organization. Strand modifications within the internucleosomal linker region are shown to involve alkaline-sensitive sites that appear to be sensitive to S1 endonuclease. Our results suggest that apoptosis is not dependent upon internucleosomal cleavage and may reveal the penultimate step and the nature of the metabolic cascade that leads to cell death.

We present theory and experimental data on the minimum detectable feature size and spatial resolution for a scanning-laser microscope system that uses bulk photodetection. In the analysis, interactions of laser photons with an object are given a probability function that varies with the position within an object. Typical interactions that can be measured with such a scanning device include photon absorption (densitometry), scattering, and photofluorescence. Because bulk photodetection is used, image resolution is a function of laser-probe spot size and recording precision. We present data from simulations that predict a minimum separation between feature centers of approximately 1.56 times the half-width of the laser spot. Experimental verification by scans of U.S. Air Force test targets confirms this theory.

Apoptosis is a process of genetically programmed alterations of cell structure that lead to failure of proliferation and differentiation, and eventual cell death. Apoptosis is induced by a variety of toxic insults including growth factor deprivation and ionizing radiation. This process may function to protect against the appearance of heritable phenotypic changes in cells and may be a critical factor in normal cellular immune function. Phorbol esters inhibit apoptosis, but little is known about factors that regulate this process physiologically. In this study, we demonstrate an association between an acute psychological stressor, taking examinations, and the induction of substantial and reversible changes in the response of peripheral blood leukocytes to gamma irradiation and to phorbol ester treatment. These data suggest that psychological stress may induce physiological changes that regulate the ability of immune cells to initiate apoptosis.

Confluent cultures of guinea pig smooth muscle cells (SMC) or human fibroblasts (HNF) were treated with 12-tetradecanoylphorbol-13-acetate (TPA). Prostanoid levels were measured by the radioimmunoassay of 6-keto-PGF1 alpha and PGE2, and lipid peroxides were measured by the thiobarbituric acid test for malondialdehyde (MDA). Cells were seeded at low densities, and growth was calculated both from the cell count (Coulter Counter) and the colony number (image analysis). When confluent SMC and HNF were incubated in media alone, 6-keto-PGF1 alpha levels were a function of the TPA concentration, increasing to a maximum at 10(-8) M TPA and then decreasing at higher TPC concentrations. When confluent SMC and HNF were incubated in media containing exogenous arachidonic acid, 6-keto-PGF1 alpha levels again increased to a maximum at 10(-8) M TPA but decreased at higher TPA concentrations only with SMC. The increase in 6-keto-PGF1 alpha levels was much greater in HNF (1310%) than SMC (680%). SMC synthesized similar amounts of 6-keto-PGF1 alpha and PGE2, and the stimulatory effect of TPA was similar with 6-keto-PGF1 alpha and PGE2. Indomethacin (IM) blocked prostanoid synthesis at all TPA concentrations. TPA did not have a significant effect on MDA levels in either cell line. The lipid antioxidants alpha-tocopherol and alpha-tocopherylquinone blocked lipid peroxidation without affecting the stimulation of prostanoid synthesis with TPA. Cell number decreased to a minimum at 10(-8) M TPA in both cell lines. The decrease in cell number was much greater in HNF (72%) than SMC (30%). SMC colony number decreased at 10(-8) TPA and then increased at 10(-6) M TPA. IM did not block the TPA effect on cell number in either cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Substantial evidence has implicated Epstein-Barr virus (EBV) in the aetiology of two human neoplasms, nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma. This is supported by the presence of high antibody titres to EBV early antigen and virus capsid antigen, as well as antibody to two viral-associated enzymes, DNase and DNA polymerase. Patients with NPC, particularly the undifferentiated form, are commonly found to have EBV DNA in the tumour. Ito and others have presented strong epidemiological evidence that phorbol esters are related to the unusual geographic distribution of NPC in southeastern regions of China. There appears to be a close link between the widespread EBV infection of the Asian population and the distinct regional distribution in China of plants that produce diterpene ester. Naturally occurring phorbol esters are produced by plants of the Euphorbiaceae and Thymelaeaceae, which are used as traditional herbal medicines. Although it has been established that EBV can infect epithelial cells isolated from NPC as well as certain normal epithelial cells, there has been no in vitro evidence that EBV induces neoplastic transformation in normal human epithelial cells with or without exposure to phorbol esters. We report here evidence that transformation of normal human epithelial cells results from exposure to infectious EBV and that transformation is dependent on the presence of phorbol esters.

The introduction of either PGF2 alpha-(10(-7) M) or TPA (10(-7) M) stimulated, ouabain-sensitive 86Rb+ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF2 alpha at these concentrations stimulated the incorporation of 3H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na+/K+ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-10T1/2 cells there were parallel increases in 3H-TdR incorporation and ouabain-sensitive 86Rb+ influxes with 10(-7) M TPA, whereas PGF2 alpha stimulated a significant increase in 3H-TdR incorporation in 3T3-4 but not C3H-10T1/2 cells and only marginal increases in ouabain-sensitive 86Rb+ influx in both. Therefore, although there appears to be a close correlation between Na+/K+ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF2 alpha cannot be confirmed.

TPA stimulates cell cycle activation in both serum-deprived and density-inhibited cultures. The cells reestablish cycle arrest after no more than one generation, and addition of fresh drug produces no further response. However, cells freshly trypsinized can respond with a series of repetitive generations resulting in 3.5-4.0 population doublings over 72 hrs. In kinetic pulse experiments TPA enhanced 3H-thymidine incorporation in density-inhibited cells stimulated by fresh serum but only after markedly suppressing incorporation 8-13 hrs after serum stimulation. When cells arrested by serum deprivation were pretreated with TPA, fresh serum stimulation led to initiation of 3H-TdR incorporation 5 hrs earlier than untreated controls. However, TPA addition at the time of serum stimulation did not lead to a suppression at 8-13 hrs, whereas enhancement was observed during peak incorporation times regardless of whether the cells were pretreated with TPA during serum deprivation. The results support the concept that there can exist within G1 multiple states of responsiveness to phorbol esters. These pharmacologically induced states may be correlated with corresponding physiological states of the G1 phase of cell cycle.

The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent.

Fifteen malignant melanomas were subjected to chromosomal analysis, in order to determine whether the number of chromosomes in a tumor cell could be correlated to a clinical prognosis. The protocol involved a direct technique which utilized a Colcemid blockade of spindle formation in mitosis to allow study of metaphase chromosomes. The direct method was chosen to reveal chromosome changes in the tumor cell in situ rather than changes in the cultured cell. Two tumors yielded chromosome spreads which could be counted and correlated with a clinical prognosis. Failure to obtain other adequate chromosome spreads were accounted for by the presence of tissue necrosis and the absence of viable tumor cells.

Serum deprivation of C3H 10T 1/2 fibroblasts resulted in DNA fragmentation which was prevented by growth factors such as Epidermal Growth Factor or the tumor promoters, 12-0-tetradecanoyl-13-0-phorbol acetate and Dihydroteleocidin B. Palmityl carnitine, an inhibitor of Ca2+-phospholipid-dependent protein kinase C, reversed the effects of the tumor promoters, but not the effect of Epidermal Growth Factor.

The expression of growth control and morphological transformation was studied in methylcholanthrene-transformed C3H/10T 1/2 CL8 cells serially propagated in CDM by first exposing cells to albumin (0.1%) before dispersing them with trypsin (50 microgram/ml). In serum-supplemented media, methylcholanthrene-transformed C3H/10T 1/2 CL8 cells exhibit various aspects of the transformed phenotype such as irregular morphology, extensive cell overlap, lack of density-dependent inhibition of division, a saturation density of 1.1 X 10(5) cells/sq cm and tumorigenicity in vivo. Cell phenotype in CDM was dramatically altered. Methylcholanthrene-transformed C3H/10T 1/2 1/2 CL8 cells adapted to CDM exhibited a regular epithelioid morphology with no cell overlap and formed confluent monolayers of nonproliferating cells at a saturation density of 5 X 10(4) cells/sq cm. The mean generation time of logarithmic-phase cells was 25 to 27 hr. Reversion to the transformed phenotype followed addition of albumin (0.1%) or serum (2%) to logarithmic-phase cultures or exposure (30 to 60 sec) to trypsin (10 microgram/ml). Cultures in CDM reexposed to serum remained highly tumorigenic in vivo. The data suggest that absorbed proteins may block transformation-sensitive cell surface sites responsible for growth control and that these sites are inactivated by trypsin.

The influence of beta-adrenergic antagonists (propranolol, pronethalol, alprenolol, isopropylmethoxamine, H 35/25, sotalol and practolol) on isotonic contractile responses to norepinephrine (NE) was studies. All drugs caused an increase in the maximum responses while depressant effects were seen only with high doses of propranolol, pronethalol and alprenolol. The enhancement of responses to NE was considerably greater at low concentrations of calcium (0.5-1.0 mM) than at high (8 mM) concentrations. The inhibitory effects of propranolol, pronethalol and alprenolol were diminished but not completely overcome by increasing calcium concentrations form 1.8 to 8 mM. Cumulative dose-response curves of calcium showed no increase in maximum responses although responses to low concentrations of calcium were augmented by sotalol and practolol. Evidence suggests that the enhancing effects of these drugs may be due to their facilitatory effect on calcium mobilization following alpha-adrenoceptor activation while their depressant properties probably reflect their membrane stabilizing properties.

The role of tumor-promoter-induced Na+, K+-ATPase activity in cell proliferation and the extent of coupling of Na+ and K+ movements to cell-cycle control under differing physiological states was examined. Earlier studies indicated that staging of cells in G1 by serum deprivation in the presence of phorbol esters such as phorbol 12-myristate 13-acetate (PMA) induced a state(s) in which postconfluent C3H 10T1/2 fibroblasts were refractory to ouabain inhibition of DNA synthesis, and the present study further examines this property. Previous findings suggested that the promoter can act in either of two ways: (i) it can act by inducing an alternative pathway to S-phase independent of Na+,K+-ATPase activity, or (ii) the promoter can advance the cells in G1 to a point beyond which Na+, K+-ATPase activity is no longer required for the induction of DNA synthesis. When ouabain (0.3 mM) was added simultaneously with tumor promoters, such as dihydroteleocidin B, to cells arrested in the G1 phase, [3H]thymidine incorporation was inhibited greater than 90%. These data suggest that an alternative pathway is not likely the explanation but that tumor promoters advance cells through a dynamic state in G1, during which progression toward S-phase entry is independent of a Na+,K+-ATPase dependent regulatory step. Ouabain sensitivity kinetics measured by two independent methods indicated that the development of ouabain insensitivity is found in G1 approximately equal to 2 hr prior to S phase. This study indicates that the measured ion movements are markedly dependent on cell-cycle state and describes the criteria required to obtain reproducible responses.