Biomedical subjects

G M Cohen

Publications and source records attributed to G M Cohen.

At least 73 records · Page 4Linked to original sources

PubMed1997

Downregulation of Bcr-Abl in K562 cells restores susceptibility to apoptosis: characterization of the apoptotic death.

We examined the susceptibility of a variety of human leukemic cell lines to the induction of apoptosis. K562, a chronic myelogenous leukemic cell line which expresses the bcr-abl fusion gene, was found to be extremely resistant to apoptosis, irrespective of the inducing agent. This resistance can be attributed to the deregulated Abl kinase activity of bcr-abl, as downregulation of its expression using antisense oligodeoxynucleotides targeted to the beginning of the abl sequence in this chimeric gene rendered these cells susceptible to cytotoxic drug-induced apoptosis. Examination of the morphological and biochemical features of apoptosis in K562 cells revealed the typical membrane blebbing and chromatin condensation associated with this form of cell death. In situ TdT-mediated end labeling of the DNA revealed the presence of strand breaks in the treated cells and field inversion gel electrophoresis revealed the presence of large 10-50 kb fragments. However there was an absence of oligonucleosomal DNA fragmentation, whether or not Bcr-Abl was expressed. Thus, while inhibition of expression of Bcr-Abl renders K562 cells susceptible to apoptosis, the absence of oligonucleosomal DNA fragmentation in these cells is independent of the function of this molecule.

Journal Article↗

PubMed1997

Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK).

N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK), a chymotrypsin-like serine protease inhibitor, affected apoptosis in human monocytic THP.1 cells differently dependent on both the concentration used and the apoptotic stimulus. TPCK (50 - 75 microM) induced both biochemical and ultrastructural changes characteristic of apoptosis, including proteolysis of poly (ADP-ribose) polymerase (PARP) and lamins together with formation of large kilobase pair fragments of DNA, particularly of 30 - 50 and 200 - 300 kilobase pairs in length but without internucleosomal cleavage of DNA. The induction of apoptosis by TPCK also involved the processing of CPP32 and Mch 3 to their catalytically active subunits. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), an ICE-like protease inhibitor, completely prevented all the biochemical and morphological changes induced by TPCK demonstrating the involvement of ICE-like proteases in the execution phase of apoptosis. Lower concentrations of TPCK (5 - 20 microM) prevented internucleosomal cleavage of DNA induced by other apoptotic stimuli. TPCK (10 microM) inhibited cell death induced by etoposide but potentiated that induced by cycloheximide demonstrating that it differentially affected apoptosis in THP.1 cells dependent on the stimulus used. These results are consistent with at least three distinct TPCK targets, one being important for cell survival, the second in facilitating internucleosomal cleavage of DNA and the third in the modulation of apoptosis induced by different apoptotic stimuli.

Journal Article↗

PubMed1997

Determination of glucose concentrations in an aqueous matrix from NIR spectra using optimal time-domain filtering and partial least-squares regression.

We have investigated the use of a time-domain optimal filtering method to simultaneously minimize both the baseline variation and high-frequency noise in near-infrared (NIR) spectrophotometric absorption data of glucose dissolved in a simple aqueous (deionized water) matrix. By coupling a third-order (six-pole) digital Butterworth bandpass filter with partial least-squares (PLS) regression modeling, glucose concentrations were determined for a set of test data with a standard error of prediction (SEP) of 10.53 mg/dl (mean percent error: 4.24%) using seven PLS factors. Compared to the unfiltered test data for six PLS factors and a SEP = 17.00 (mean percent error: 7.38%) this results shows more than a 38% decrease in the error. The glucose concentrations ranged from 51 mg/dl to 493 mg/dl, and the NIR spectral region between 2088 nm and 2354 nm (4789 cm-1 and 4248 cm-1) was used to develop the optimal PLS model. The optimal PLS model was determined from a sequence of three-dimensional performance response maps for different numbers of PLS factors (2-10). A total of 99 NIR spectra were generated for glucose dissolved in deionized water using a NIRsystems 5000 dispersive spectrophotometer. Nine of these spectra were generated for only water, which were averaged and subtracted from the remaining 90 spectra to generate the training and test data sets, thereby, removing the intrinsic high background absorption due to the water. The training set consisted of 57 spectra and associated glucose concentration target values, and the test set was comprised of the remaining 33 spectra and target values. Performance results were compared for three different digital Butterworth bandpass filters (four-poles, six-poles, and eight-poles), and a digital Gaussian filter design approach (i.e., Fourier filtering).

Blood Glucose Self-Monitoring↗

PubMed1996

Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32.

Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.

Amino Acid Chloromethyl Ketones↗

PubMed1996

A cleavage-site-directed inhibitor of interleukin-1 beta-converting enzyme-like proteases inhibits apoptosis in primary cultures of rat hepatocytes.

Apoptosis induced in primary hepatocytes by transforming growth factor beta1 and staurosporine produced chromatin condensation, DNA cleavage is detected by in situ end-labelling, field inversion and conventional gel electrophoresis, and cell detachment. These effects were abolished by benzyloxycarbonyl-valinylalanylaspartylfluoromethyl ketone, a cleavage-site-directed inhibitor of interleukin-1beta-converting enzyme-like proteases, and this finding suggests that these enzymes are involved in liver apoptosis.

Alkaloids↗

PubMed1996

Multivariate determination of glucose concentrations from optimally filtered frequency-warped NIR spectra of human blood serum.

Glucose concentrations over the 39-160 mg dl-1 range have been determined from 357 NIR (near-infrared) spectra of human blood serum in the spectral region from 6766 cm-1 to 4003 cm-1. A frequency-warping procedure was applied to the NIR data to compress 511 spectral components into 102 in the 6766-4003 cm-1 spectral region. Before the data compression process was carried out, the NIR spectrum of deionized water was subtracted from each of the blood serum spectra to remove the intrinsic high background absorption due to the water. PLS (partial least-squares) regression was coupled with time-domain digital Butterworth bandpass filtering in an optimization procedure. The optimization procedure was carried out over a range of centre frequencies and bandwidths for first- (two-pole), second- (four-pole) and third- (six-pole) order bandpass filters, and over a range of PLS factors. The optimal PLS model and filter parameters were determined from a sequence of three-dimensional performance response maps for different numbers of PLS factors and filter orders. As a basis for comparison, the same optimization process was carried out for a Gaussian filter design approach (i.e., Fourier filtering). Using the optimally filtered frequency-warped NIR spectral data, an SEP (standard error of prediction) of 13.2 mg dl-1 was achieved fro the test (monitoring) data using 14 PLS factors and a simple first-order (two-pole) digital Butterworth bandpass filter.

Blood Glucose↗

PubMed1996

Acid phosphatase activity in the chick's inner ear.

The cellular localization of distribution of acid phosphatase (AP) in the plastic sections of newly hatched chick's inner ear were investigated utilizing an azo-coupling method. AP activity as evidenced by azo dye deposits were well defined and localized in various cells of membranous labyrinth. Intense AP activity was detectable in the supranuclear area of hair cells in the basilar papilla and vestibular sensory hair cells. As in the case of the other sites of AP activity, marked AP activity was seen in the supranuclear area of the transitional epithelia of crista ampullaris and in the supranuclear area, or diffusely in the cytoplasm of the dark cells at the base of crista ampullaris. The columnar cells and the cells of tegmentum vasculosum showed moderate to strong AP activity. The statoacoustic and vestibular ganglion cells showed various degrees of AP activity. On the AP activity of statoacoustic or vestibular ganglion, in comparison between the sections from JB-4 Plus embedded specimens and those from LR White embedded specimen, the latter could more intensely demonstrate AP activity than the former. Moreover, sections fixed in 2.5% paraformaldehyde demonstrated more intense AP activity than those fixed in 2.5% glutaraldehyde.

Acid Phosphatase↗

PubMed1996

DNA-damaging agents induce both p53-dependent and p53-independent apoptosis in immature thymocytes.

Apoptosis in the immature thymus can be induced by both p53-dependent and -independent pathways, the former being activated by exposure to DNA-damaging agents and the latter being induced by glucocorticoids [Nature (Lond.) 362:847-849; Nature (Lond.) 362:849-852 (1993)]. We report that the DNA-damaging agents etoposide and gamma-radiation induced similar levels of apoptosis in both proliferatively enriched and quiescent immature rat thymocytes, as assessed by flow cytometry and the formation of both kilobase-pair and 180-bp integer fragments of DNA. However, a marked stabilization of p53 occurred exclusively in the proliferatively enriched population, which was also enriched for immature CD4- CD8- and mature CD4+ CD8-/CD4- CD8+ cells. In contrast, DNA damage-induced apoptosis in quiescent mature peripheral T cells was associated with an accumulation of p53. Our studies suggest that stabilization of p53 in thymocytes in response to DNA damage may be developmentally regulated. In immature thymocytes obtained from p53-null mice, DNA-damaging agents induced apoptosis at significantly lower levels and at later times than that seen in cells from p53 wild-type animals. These data support the hypothesis that DNA-damaging agents induce apoptosis primarily via a p53-dependent pathway in immature thymocytes as previously reported. We report here that DNA damage can also induce apoptosis by a p53-independent pathway in a particular subpopulation of immature thymocytes.

Amino Acid Sequence↗

PubMed1995

An interleukin-1 beta-converting enzyme-like protease is a common mediator of apoptosis in thymocytes.

Apoptosis was induced in thymocytes using diverse stimuli in order to identify events within a common apoptotic pathway. Benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone (Z-VAD.FMK), an interleukin-1 beta-converting enzyme (ICE)-like protease inhibitor, inhibited apoptosis assessed by flow cytometry, proteolysis of poly (ADP)-ribose polymerase of DNA to both large kilobase pair fragments (30-50 and 200-300 kbp) and to nucleosomal fragments. Z-VAD.FMK also blocked all the classical ultrastructural features of apoptosis including chromatin condensation to one pole of the nucleus, nucleolar disintegration and cytoplasmic vacuolation. These results suggest the involvement of an ICE-like protease as a common mediator of apoptosis in thymocytes.

Amino Acid Chloromethyl Ketones↗

PubMed1995

An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells.

Apoptosis was induced in THP.1 cells, a human monocytic tumour cell line, by diverse stimuli including cycloheximide, thapsigargin, etoposide and staurosporine. Induction of apoptosis by all these stimuli, except etoposide, was enhanced in the presence of the trypsin-like protease inhibitor, N alpha-tosyl-L-lysinyl chloromethyl ketone (TLCK). Induction of apoptosis, assessed by morphological, flow cytometric and biochemical criteria, including proteolysis of poly(ADP-ribose) polymerase and cleavage of DNA to large kilobasepair fragments, was completely abrogated when cells were pretreated with an ICE-like protease inhibitor, Z-Val-Ala-Asp.fluoromethylketone. This suggested that an ICE homologue was a common mediator of apoptosis in THP.1 cells.

Amino Acid Chloromethyl Ketones↗

PubMed1995

Cleavage of DNA to large kilobase pair fragments occurs in some forms of necrosis as well as apoptosis.

In a number of cellular systems, DNA in apoptotic cells in initially degraded into large fragments of 30-50, 200-300 and > or = 700 kilobase pairs, which may subsequently give rise to oligonucleosomal fragments often considered as the biochemical hallmark of apoptosis. In this study, necrosis was induced in U937 cells by incubation with water. Cells yielded large DNA fragments similar in size to those found in apoptosis, but the DNA was then degraded to a continuous spectrum of small fragments, confirming that death was necrotic. The results demonstrate that kilobase pair DNA fragments are formed in some instances of necrosis as well as in apoptosis. This indicates that apoptosis should not simply be assessed by the formation of kilobase pair DNA fragments, but that other criteria, such as cellular morphology, must also be used to verify apoptosis.

Apoptosis↗

PubMed1995

Multi-step DNA cleavage in rat liver nuclei is inhibited by thiol reactive agents.

DNA fragmentation in isolated rat liver nuclei is a Mg(2+)-dependent, multi-step process which is potentiated by Ca2+ and cleaves the DNA into > or = 700, 200-300 and 30-50 kilobase pair (kbp) fragments, prior to internucleosomal cleavage by Ca2+/Mg(2+)-dependent endonuclease(s). We now show that Cd2+, Hg2+, dichloroisocoumarin (DCI, a serine protease inhibitor) and N-ethylmaleimide (NEM) block both Mg2+ and Ca2+/Mg(2+)-dependent processes. Inhibition of DNA cleavage produced an increase in the size of the DNA fragments, from mono-/oligonucleosomes to 30-50, 200-300, > or = 700 kbp and finally to intact DNA. NEM and DCI inhibition was blocked by dithiothreitol, and it is proposed that a critical thiol(s) is involved in the DNA cleavage reactions which are a feature of the apoptotic process.

Animals↗

PubMed1995

A pre-existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli.

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the inhibitors N-tosyl-L-lysyl chloromethylketone (TLCK) and N-tosyl-L-phenylalanyl chloromethylketone (TPCK) have very different effects on apoptosis. TLCK inhibits apoptosis induced by diverse stimuli at an early stage prior to both DNA fragmentation and cytoplasmic changes. We show that the TLCK-sensitive target is pre-existing and not synthesized in response to apoptotic stimuli. The contrasting effects of TLCK and TPCK support the hypothesis that the TLCK target is a trypsin-like protease which is a common effector of thymocyte apoptosis.

Animals↗

PubMed1995

DNA degradation and proteolysis in thymocyte apoptosis.

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethylketone (Z-VAD.FMK) inhibit apoptosis. N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) has a very different effect, inducing the early morphological and biochemical changes associated with apoptosis. TLCK inhibits trypsin-like proteases whilst Z-VAD.FMK inhibits interleukin-1 beta-converting enzyme (ICE)-like proteases; this and the contrasting effects of TPCK support the hypothesis that thymocyte apoptosis involves a hierarchy of proteases which act at different stages of the process.

Animals↗

PubMed1994

The comparative toxicity of chlorambucil and chlorambucil-spermidine conjugate to BALB/c mice.

The acute intraperitoneal toxicities of chlorambucil and chlorambucil-spermidine conjugate have been compared, in mice. Both compounds were neurotoxic and also caused a prolonged fall in bodyweight and a depletion of lymphocyte numbers associated with a fall in the total leukocyte count and loss of spleen and thymus weight. Alanine aminotransferase and aspartate aminotransferase activities and blood urea nitrogen concentration were increased at 24 h after conjugate administration, but had returned to normal at 72 h. Chlorambucil significantly decreased blood urea nitrogen concentration for 72 h, but did not affect transferase activity. Tissue concentrations of conjugate were measurable in liver and kidney for 12 days and lung for 5 days after dosing. The toxicity of both compounds was cumulative. In mol/kg, the chlorambucil-spermidine conjugate was 10-fold more toxic than chlorambucil, on the basis of their neurotoxicity, but only 2- to 3-fold more toxic on the basis of their effects on lymphocyte depression. The increased toxicity of the conjugate does not improve its therapeutic index relative to chlorambucil.

Alanine Transaminase↗

PubMed1994

A role for metals and free radicals in the induction of apoptosis in thymocytes.

Recent reports have implicated a possible but undefined role for reactive oxygen species in the induction and mediation of apoptosis. In the present study, the role of free radicals and metal ions in apoptosis induced in rat thymocytes by dexamethasone and etoposide was examined. Copper chelators, but not iron specific chelators, inhibited apoptosis induced by both these stimuli. Several antioxidants also possessed potent inhibitory effects. We therefore propose that diverse agents may induce apoptosis in thymocytes by a common mechanism involving a copper mediated Fenton reaction, generating site specific hydroxyl radicals, possibly as a result of activation of the redox sensitive transcription factor NF-kappa B.

Animals↗

PubMed1994

Dexamethasone and etoposide induce apoptosis in rat thymocytes from different phases of the cell cycle.

Dexamethasone and etoposide both induce apoptosis in immature rat thymocytes. We investigated the dependence of apoptosis on the phase of the cell cycle after incubation with these drugs. Cell cycle progression was followed by a combination of pulse labelling with 5-bromo-2'-deoxyuridine (BrdU), labelling fixed cells with an anti-BrdU antibody and flow cytometry. Dexamethasone had little effect on the cell cycle progression of proliferating thymocytes, while etoposide caused cell cycle arrest. Normal and apoptotic thymocytes were separated by centrifugation on discontinuous Percoll gradients into four fractions (F1-F4). It was found that both dexamethasone and etoposide induced apoptosis in cells in G0/G1 and G2/M of the cell cycle, whereas only etoposide induced apoptosis of cells in S phase. These results demonstrated that dexamethasone induced apoptosis in quiescent cells while only etoposide could induce apoptosis in cells from the proliferative compartment. Following treatment of thymocytes with etoposide, some of the proliferating thymocytes (F1) were converted to cells with intermediate size and density (F3). We have recently identified these cells as a population of preapoptotic thymocytes, at an early stage of apoptosis. These cells then further progressed to fully apoptotic cells (F4). These data support the hypothesis that normal thymocytes (F1) became apoptotic (F4) via an intermediate population (F3).

Animals↗

PubMed1994

Cdc2 activation is not required for thymocyte apoptosis.

Apoptosis is a mode of cell death characterized by chromatin condensation and disassembly of the nuclear lamina, processes that are also characteristic of mitosis. The apparent similarity between the two events, together with observations that apoptosis can occur following G2 arrest, has led to the suggestion that apoptosis could be a defective form of mitosis. Further support for this idea comes from the recent description of activation of the p34cdc2 protein kinase (Cdc2), the universal M-phase promoter, during death induced by a lymphocyte granule protease. We have monitored the protein kinase activity of Cdc2 during apoptosis of primary rat thymocytes, which die from a quiescent (G0) state. We demonstrate unequivocally that activation of Cdc2 is not involved in the induction of apoptosis in thymocytes, indicating that chromatin condensation and lamina disassembly occur in this system by processes different from those that operate in mitosis.

Animals↗