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C Dive

Publications and source records attributed to C Dive.

At least 37 records · Page 2Linked to original sources
PubMed1998

The non-genotoxic hepatocarcinogen nafenopin suppresses rodent hepatocyte apoptosis induced by TGFbeta1, DNA damage and Fas.

The suppression of apoptosis may contribute to the carcinogenicity of the peroxisome proliferators (PPs), a class of non-genotoxic rodent hepatocarcinogens. Our previous work demonstrated that the PP nafenopin suppressed both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced hepatocyte apoptosis both in vivo and in vitro. Here, we extend these observations by demonstrating the ability of nafenopin to suppress apoptosis induced by other major candidates for the signalling of cell death in the liver. Treatment of rat or mouse hepatocyte monolayers with TGFbeta1 or the DNA damaging drugs etoposide or hydroxyurea induced high levels of apoptosis. Western blot analysis did not support a role for either p53 or p21waf1 in etoposide-induced apoptosis in rat hepatocytes. Treatment of mouse hepatocytes with an agonistic anti-Fas antibody also resulted in an induction of high levels of apoptosis. Pre-addition and continued exposure to nafenopin suppressed apoptosis induced by all three stimuli. Overall, our studies demonstrate that the ability of nafenopin to protect hepatocytes from apoptosis is not restricted to species or apoptotic stimulus. It is possible, therefore, that the PPs may suppress apoptosis by acting on diverse signalling pathways. However, it seems more likely that nafenopin suppresses hepatocyte apoptosis elicited by each death stimulus by impinging on a core apoptotic mechanism.

Animals↗
PubMed1998

Cisplatin- and paclitaxel-induced apoptosis of ovarian carcinoma cells and the relationship between bax and bak up-regulation and the functional status of p53.

We investigated the roles of p53 and Bcl-2 homologues in the induction of apoptosis by cisplatin and paclitaxel in wild-type p53-expressing human ovarian carcinoma cells and cisplatin-resistant derivatives that have lost p53 function. Cisplatin induced apoptosis in parental A2780 but not in cisplatin-resistant A2780/cp70 cells, whereas paclitaxel induced apoptosis in both cell lines. Immunoprecipitation of p53 using antibodies specific for p53 conformation (pAb 1620 and pAb 240) showed that there were no relative changes in p53 conformation before and after cisplatin treatment in either cell line. A2780/cp70 cells have lost p53 function, yet they have wild-type p53 gene sequence. However, A2780/cp70 cells constitutively express more p53 in a form detected by pAb 240, an antibody that also detects mutant conformations of p53 that are transcriptionally inactive. There were no changes in levels of Bcl-2, Bcl-XL, or 24-kDa Bax over 72 hr after exposure to cisplatin or paclitaxel, but each agent led to up-regulation of Bak and 21-kDa Bax in A2780 cells. Paclitaxel, but not cisplatin, increased Bak and 21-kDa Bax levels in A2780/cp70 cells. These data suggest that apoptosis in A2780 and A2780/cp70 is associated with an increased level of Bak and 21 kDa Bax after drug-induced damage and that functional p53 may be required for this effect after cisplatin but not after paclitaxel.

Antineoplastic Agents↗
PubMed1997

Germinal center-derived signals act with Bcl-2 to decrease apoptosis and increase clonogenicity of drug-treated human B lymphoma cells.

Bcl-2 suppresses drug-induced apoptosis in vitro, although in many cases, this results only in a delayed onset of cell death. In vivo survival signals from the extracellular environment may also contribute to drug resistance and may act with Bcl-2 to promote long-term cell survival. Ligation of CD40 on B-lymphocytes in germinal centers (GCs) can suppress apoptosis induced by calcium ionophore or anti-IgM in vitro. We asked whether a combination of Bcl-2 expression and the provision of a culture environment that mimicked that of the GC [CD40 ligation and interleukin 4 (IL-4)] could increase the ability of B lymphoma cells to resist drug-induced apoptosis. A Burkitt lymphoma (BL) cell line transfected with either human bcl-2 (BL-bcl-2) or control plasmid (BL-Sv2) was used to examine the effects of Bcl-2 overexpression on the cellular response and long-term survival after treatment with the DNA-alkylating drug chlorambucil (CMB) in the presence or absence of CD40 ligation and IL-4. Administration of 20 microM CMB completely prevented cell proliferation. This was associated with an increase in p53 protein levels within 24 h, without an elevation in p21, Bax, or Mdm2 proteins. Analyses of cell cycle distribution and of cyclin B expression demonstrated that both cell lines arrested at G2/M, where they died. Fifty % of BL-Sv2 cells died within 2 days, whereas 50% cell death was not observed in the BL-bcl-2 cultures until 6 days had passed. Cross-linking of CD40 with a monoclonal antibody elevated Bcl-xL protein levels by 3 h and also provided a delay in CMB-induced death. Ninety-six h after the addition of 20 microM CMB, 78% of the BL-Sv2 cells were apoptotic, whereas ligation of CD40 on BL-Sv2 cells reduced the proportion of apoptotic cells to 38%. Overexpression of Bcl-2 (in BL-bcl-2 cells) reduced apoptosis to 41%. However, when the BL-bcl-2 cells were treated with CMB together with ligation of CD40, apoptosis was reduced further to only 17% at 96 h. The Bcl-2-mediated delay in the execution of CMB-induced apoptosis did not translate significantly to increased clonogenicity. In contrast, the provision of BL-Sv2 cells with an ability to interact with the adhesion molecule vascular cell adhesion molecule-1, CD40 ligation, and IL-4 significantly increased clonogenic survival, and this was improved in BL-bcl-2 cells exposed to these GC-derived signals. These data demonstrate that the kinetics of drug-induced apoptosis can be modulated by Bcl-2 as well as by IL-4, vascular cell adhesion molecule-1, and CD40 ligation, the latter possibly involving the function of Bcl-xL. That these factors appear to act together to enhance proliferative potential after DNA damage has important implications regarding the development of drug resistance in B-cell lymphomas and future strategies for improved chemotherapy.

Antineoplastic Agents, Alkylating↗
PubMed1997

v-Abl protein tyrosine kinase (PTK) mediated suppression of apoptosis is associated with the up-regulation of Bcl-XL.

We demonstrated previously that the activation of v-Abl protein tyrosine kinase (PTK) in IC.DP murine pre-mast cells resulted in suppression of apoptosis after withdrawal of interleukin 3 (IL-3), that protein kinase C (PKC) translocated to the nucleus 6 h after v-Abl PTK activation and that inhibition of PKC restored apoptosis after IL-3 deprivation in the presence of v-Abl PTK activity. Here we demonstrate that v-Abl PTK activation is followed by an approximately twofold increase in mRNA level of Bcl-XL by 6 h and a corresponding increase in Bcl-XL protein level by 24 h. Bcl-xL RNA and protein decreased in IL-3 deprived cells in the absence of v-Abl PTK activity. Exposure of cells with v-Abl PTK active to the PKC inhbitor calphostin C (125 ng/ml) prevented the increase in Bcl-xL protein and resulted in apoptosis. No changes in Bax or Bcl-2 protein level were noted after IL-3 withdrawal and/or activation of v-Abl PTK. Bak was barely detectable and Bad protein level decreased in cells undergoing apoptosis. The data suggest that suppression of apoptosis by v-Abl PTK in the absence of IL-3 is associated with PKC signalling and the upregulation of Bcl-xL in IC.DP cells.

Animals↗
PubMed1997

Role of acid/base homeostasis in the suppression of apoptosis in haemopoietic cells by v-Abl protein tyrosine kinase.

Removal of interleukin-3 from murine IC.DP pre-mast cells results in irreversible commitment to apoptosis within 18 hours. To identify early events necessary for the engagement of apoptosis we examined the regulation of intracellular pH (pH(i)). IC.DP cells acidified 2 hours after removal of interleukin-3 (before discernible signs of apoptosis) and by 18 hours pH(i) had decreased by 0.15 units. The acidification was due to both an increase in an acid-loading process which only occurs when intracellular pH is above 6.8 and a slight reduction in H+ efflux via NA+/H+ exchange. Activation of a temperature sensitive mutant of v-Abl protein tyrosine kinase suppressed apoptosis of IC.DP cells in the absence of interleukin-3 but did not stimulate proliferation, and moreover prevented cellular acidification. Acidification of the cells by 0.2 units to pH 6.86 by complete inhibition of Na+/H+ exchange by 10 microM 5'-(N-methyl-N-isobutyl)-amiloride prevented the suppression of apoptosis by v-abl protein tyrosine kinase following IL 3 withdrawal. However in the presence of interleukin-3, addition of 10 microM 5'-(N-methyl-N-isobutyl)-amiloride only resulted in a fall of pH(i) to 7.17. Apoptosis did not occur and the cells continued to proliferate. Thus, in this model intracellular pH must fall below a critical value for apoptosis to occur. Together these data point to a step in cytokine deprivation induced apoptosis (at least in some haemopoietic cell types) which is either enhanced by or dependent upon an acidic intracellular environment which is the result of an increase in acid loading and inhibition of Na+/H+ exchange activity. One of the mechanisms by which activation of v-Abl protein tyrosine kinase suppresses apoptosis is by prevention of intracellular acidification.

Acid-Base Equilibrium↗
PubMed1997

Avoidance of apoptosis as a mechanism of drug resistance.

Inherent or acquired drug resistance is a major obstacle for the successful treatment of cancers. Many mechanisms of drug resistance have been described including a decreased drug uptake, an increase in DNA damage repair, enhanced drug detoxification, an altered level or mutation of the intracellular drug target or an increased drug efflux from the cell. Most of these mechanisms impinge upon the interaction of a drug with its cellular target or immediate consequences of such as interaction. For example, a decrease in the cellular levels of topoisomerase II thwarts the efficacy of certain topoisomerase II inhibitors, and enhanced levels of glutathione increase resistance to DNA alkylating agents. However, some tumours are inherently resistant to all chemotherapeutic agents, i.e. with different mechanisms of action. What is the mechanism(s) underlying this pleiotropic drug resistance? One possibility is that such drug-resistant tumour cells have an abnormally high threshold for the engagement of apoptosis (programmed cell death). The suppression of apoptosis as a mechanism for drug resistance is discussed in this article.

Apoptosis↗
PubMed1996

Characterization of cell volume loss in CEM-C7A cells during dexamethasone-induced apoptosis.

A reduction in cell volume is a fundamental feature of apoptosis. We have characterized changes in cell volume, together with nuclear changes, occurring in dexamethasone-induced apoptosis in CEM-C7A lymphoblastoid cells. Cell volume was measured by electronic cell sizing and flow cytometry, and two distinct phases of volume loss were observed. The first phase began 12 h after addition of dexamethasone (5 microM) and progressed until 36 h when chromatin condensation was detected in intact cells. Removal of dexamethasone before 36 h (the precommitment period) resulted in reversal of the volume decrease and prevented the appearance of nuclear changes. Cell shrinkage in the first 24 h of dexamethasone exposure was associated with a net loss of potassium but no change in cellular buoyant density. There were no significant differences in the rates of volume recovery after either hypertonic or hypotonic stimuli. These observations favor a mechanism of cell shrinkage involving loss of the entire cytoplasmic contents, possibly following proteolysis, rather than loss of only osmolytes and water. The second phase of volume loss was coincident with chromatin condensation and was associated with cellular fragmentation and a reduction in cellular density. We conclude that volume loss in this model of apoptosis is mediated by multiple mechanisms that are both dependent and independent of cellular fragmentation.

Apoptosis↗
PubMed1996

Requirement of basement membrane for the suppression of programmed cell death in mammary epithelium.

Apoptosis is an active mechanism of cell death required for normal tissue homeostasis. Cells require survival signals to avoid the engagement of apoptosis. In the mammary gland, secretory epithelial cells are removed by apoptosis during involution. This cell loss coincides with matrix metalloproteinase activation and basement membrane degradation. In this paper we describe studies that confer a new role for basement membrane in the regulation of cell phenotype. We demonstrate that the first passage epithelial cells isolated from pregnant mouse mammary gland die by apoptosis in culture, but that cell death is suppressed by basement membrane. The correct type of extracellular matrix was required, since only a basement membrane, not plastic or a collagen I matrix, lowered the rate of apoptosis. Attachment to a matrix per se was not sufficient for survival, since apoptotic cells were observed when still attached to a collagen I substratum. Experiments with individually isolated cells confirmed the requirement of basement membrane for survival, and demonstrated that survival is enhanced by cell-cell contact. A function-blocking anti-beta1 integrin antibody doubled the rate of apoptosis in single cells cultured with basement membrane, indicating that integrin-mediated signals contributed to survival. We examined the cell death-associated genes bcl-2 and bax in mammary epithelia, and found that although the expression of Bcl-2 did not correlate with cell survival, increased levels of Bax were associated with apoptosis. We propose that basement membrane provides a survival stimulus for epithelial cells in vivo, and that loss of interaction with this type of matrix acts as a control point for cell deletions that occur at specific times during development, such as in mammary gland involution.

Animals↗
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↗
PubMed1996

Cisplatin, camptothecin, and taxol sensitivities of cells with p53-associated multidrug resistance.

With the use of clonogenic survival assays, we show that wild-type p53-expressing A2780 human ovarian cell lines transfected with a dominant negative mutant p53 gene (codon 143, valine to alanine) acquired cross-resistance to ionizing radiation, cisplatin, doxorubicin, and 1-beta-D-arabinofuranosylcytosine. However, these mutant p53-transfected cell lines retained sensitivity to taxol and camptothecin. We also show that immature thymocytes from mice with the p53 gene genetically inactivated showed reduced ability to undergo apoptosis after treatment with ionizing radiation and cisplatin compared with wild-type mice. However, taxol-induced apoptosis in thymocytes does not seem to be dependent on p53 status. Camptothecin also induced apoptosis in a p53-independent manner in thymocytes at low doses but in a p53-dependent manner at high doses. These data suggest that taxoids and camptothecin analogs could have activity in tumors that have aberrant p53 function and provide a rationale for the clinical observations of responsiveness of refractory ovarian cancer to these drugs.

Animals↗
PubMed1995

Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukaemic cells.

Apoptosis, originally defined by specific morphological changes, is characterised biochemically by non-random cleavage of DNA. Depending on cell type, this DNA cleavage proceeds from 300 and 50kbp fragments prior to, concomitantly with, or in the absence of 180bp integer fragmentation. Incorporation into fragmented DNA of biotin-labelled nucleotides by terminal deoxynucleotidyl transferase (TdT) has recently become a standard flow cytometric assay for the identification and quantitation of apoptosis. Nucleotide incorporation is visualized using avidin-tagged fluorescein isothiocyanate (FITC) (Gorczyca et al.: Cancer Res 53:1945-1951, 1993; Jonker et al.: Cytometry (Suppl 13):Abstr 99A, 1993). Here, we characterise this assay further in three different haemopoietic cell lines. Drug-induced DNA damage is not identified by the TdT assay unless it is coupled to the apoptotic response. This was demonstrated using cells in which activation of the oncogenic Abelson-encoded protein tyrosine kinase suppressed drug-induced apoptosis, but did not inhibit drug-induced DNA damage (by melphalan, hydroxyurea, or etoposide). Furthermore, the TdT assay identifies DNA fragments formed during apoptosis induced by etoposide and N-methylformamide in HL60 and MOLT-4 cells, including those high molecular weight DNA fragments formed in MOLT-4 cells which were not further cleaved to 180-200bp integer fragments. Our results support the use of flow cytometry and the TdT assay to reliably measure apoptotic cells in heterogeneous cell samples.

Abelson murine leukemia virus↗
PubMed1995

MCF-7 human mammary adenocarcinoma cell death in vitro in response to hormone-withdrawal and DNA damage.

Anti-oestrogens exert a tumoristatic effect on estrogen-receptor-positive breast carcinomas in vivo. At a cellular level this may reflect inhibition of cell proliferation and/or cell death counterbalanced by continued proliferation of a cell subpopulation. We evaluated the MCF-7 human mammary adenocarcinoma cell line as an in vitro model to study the effects of the novel oestrogen antagonist ICI 182,720 on cell population dynamics (cell gain vs. cell loss). After oestrogen-withdrawal monolayer cell number declined over 10 days, accompanied by cell detachment. This decrease in viable cell number was elevated 2-fold by ICI 182,780. Detached cells exhibited DNA fragments of 50 and 300 kbp, typical of apoptotic cells. However, internucleosomal cleavage to 180 bp integer fragments was not seen, and these detached cells exhibited a morphology which was not consistent with apoptosis. The remaining attached monolayer cells were morphologically viable (> 99%) with regard to both nuclear morphology and plasma membrane integrity. There was no difference in cell cycle phase distribution between oestrogen-withdrawn and ICI 182,780-treated cells; both induced accumulation in G1 phase. MCF-7 cells were also exposed to a variety of DNA damaging agents known to induce apoptosis in other cell types. We could demonstrate only limited induction of morphologically recognisable apoptosis in MCF-7 cells treated with methyl methanesulphonate. Our results add to the controversy surrounding the ability of the MCF-7 cell line to undergo apoptosis in vitro in response to anti-oestrogen therapies.

Adenocarcinoma↗
PubMed1995

Endoscopic Nd:YAG treatment of superficial gastric carcinoma: experience in 18 Western inoperable patients.

Endoscopic treatment of superficial gastric cancer has been reported to be effective by many Japanese teams. In this study, the Nd:YAG laser was used to treat superficial gastric carcinoma in inoperable Caucasian patients with the aim of obtaining a complete response, i.e., disappearance of the lesion endoscopically and biopsy specimens negative for cancer. Eighteen patients unsuitable for surgery with various endoscopic patterns of superficial gastric cancer were treated with the Nd:YAG laser. The endoscopic pattern was type I in 4 patients, type II in 10 (5 type IIa, 1 type IIb, 2 type IIc, 2 mixed IIa + IIc), and type III in 4. Staging by echoendoscopy was performed in 11 patients (T1N0). Nd:YAG laser destruction of the gastric tumor was performed in all cases, with a mean of 4.4 laser sessions per patient. Tumor response was assessed by endoscopy and biopsy. Follow-up averaged 33 +/- 23 (SD) months. Five patients died of diseases unrelated to gastric cancer. An initial complete response was obtained in 16 (89%) patients after a mean of 1.7 laser sessions; histologic evidence of cancer persisted in 2 patients during the entire follow-up period. Among patients with an initial complete response, recurrence was observed in 2. One of them was successfully re-treated. At the end of the follow-up period, 14 (77.7%) of the 18 patients had a complete tumoral response; only 4 patients had histologic evidence of cancer. In 3 of these 4 patients, pretherapeutic echoendoscopic staging had not been performed. Among the 14 patients exhibiting a complete response, 3 had negative biopsy results more than 5 years after diagnosis. No complications occurred. In gastric cancer classified as T1N0 on the basis of pretherapeutic echoendoscopy, a high tumor response rate and even 5-year disease-free survival can be obtained with endoscopic Nd:YAG laser treatment. Endoscopic laser destruction thus appears to be a valuable therapeutic alternative to surgery in inoperable patients with superficial gastric cancer.

Aged↗
PubMed1995

Differences in resistance to 5-fluorouracil as a function of cell cycle delay and not apoptosis.

A series of human embryo fibroblasts has previously been shown to display increasing resistance to the antimetabolites methotrexate (MTX) and N-phosphonacetyl-L-aspartate (PALA) with increasing tumorigenicity. This increased resistance was found to be further increased as a result of salvage pathway activity for purine and pyrimidine biosynthesis. A similar pattern of increasing resistance paralleling increasing tumorigenicity has now been shown to occur with 5-fluorouracil (5-FU), which is independent of salvage pathway activity. The KMS normal cell line was found to be more sensitive to 5-FU than either the immortalised KMST or tumorigenic KN-NM cell lines. Immunohistochemical analysis of the three cell lines demonstrated high levels of p53 protein in the KMST and KN-NM cell lines, but undetectable p53 levels in the KMS cell line. From these data it was hypothesised that a difference in p53 function may be causing the difference in the patterns of sensitivity observed in the three cell lines. P53 is now believed to function as a regulator of a G1 to S cell cycle checkpoint and as an inducer of apoptosis following DNA damage to the cell. The differences in sensitivity of the cell lines could not be explained by differences in the levels of apoptosis but could be attributed to differences in cell cycle response. Our evidence suggests that loss of cell cycle control, possibly through loss of p53 function, is an important factor in increasing the drug resistance of fibroblast cell lines.

Antimetabolites, Antineoplastic↗
PubMed1995

Suppression of apoptosis by v-ABL protein tyrosine kinase is associated with nuclear translocation and activation of protein kinase C in an interleukin-3-dependent haemopoietic cell line.

We previously demonstrated that activation of v-ABL protein tyrosine kinase resulted in suppression of apoptosis following interleukin-3 removal using an interleukin-3-dependent haemopoietic cell line transfected with a temperature-sensitive mutant of the v-abl oncoprotein (IC.DP). Cellular signalling events associated with the activation of v-ABL included increased levels of sn-1,2-diacylglycerol, an activator of protein kinase C. Calphostin C, a PKC inhibitor, restored apoptosis to interleukin-3-deprived IC.DP cells expressing active v-ABL. However, chronic exposure to the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate to downregulate protein kinase C did not attenuate the survival of IC.DP cells expressing active v-ABL. Translocation of a classical protein kinase C isozyme(s) to the nuclear fraction was observed 6 hours after activation of v-ABL, when nuclear protein kinase C activity was increased approximately 2-fold. The protein kinase C isozyme responsible, which was only partially downregulated by 12-O-tetradecanoyl phorbol 13-acetate, was identified as protein kinase C beta II. This translocation of protein kinase C beta II to the nucleus was inhibited by calphostin C. Taken together, these results suggest that nuclear translocation and activation of PKC beta II may play a role in v-ABL-mediated suppression of apoptosis.

Animals↗
PubMed1995

Characterization of drug resistance mediated via the suppression of apoptosis by Abelson protein tyrosine kinase.

Constitutive activation of the Abelson (Abl) protein tyrosine kinase (PTK) is a causative event in chronic myeloid leukemia, where intense chemotherapy currently fails to eradicate the leukemic clone. Using a mouse mast cell line (IC.DP), we previously showed that v-Abl PTK induced resistance to the anti-cancer drugs melphalan and hydroxyurea by the suppression of apoptosis. Here, using this cell line, we demonstrate by alkaline elution that v-Abl PTK did not affect the levels of DNA damage induced by either drug. This confirms that v-Abl PTK acts downstream of the drug-target interaction to prevent the coupling of drug-induced damage to the apoptotic pathway. Although Abl PTK- and interleukin-3 (IL-3)-stimulated signaling events share common signaling pathways, a similar level of drug resistance was not provided by IL-3, implying that Abl PTK does not merely mimic an IL-3 survival signaling pathway. Previously we demonstrated translocation of protein kinase C-beta II stimulated by activation of Abl PTK. Drug sensitivity was restored in cells with active v-Abl PTK by simultaneous addition of calphostin C, an inhibitor of protein kinase C, suggesting a role for protein kinase C in the suppression of drug-induced apoptosis by v-Abl PTK. One novel strategy for the treatment of chronic myeloid leukemia could therefore include the use of a downstream modifier of the Abl PTK-mediated survival signaling pathway to render leukemic cells more sensitive to a second drug, such as a cytotoxic agent.

Abelson murine leukemia virus↗
PubMed1995

Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis.

Apoptosis is characterized by the nonrandom cleavage of DNA. After continuous treatment of MOLT-4 human T lymphoblastoid cells with the topoisomerase II inhibitor etoposide (50 microM) and the nongenotoxic agent N-methylformamide (300 mM), apoptosis was confirmed by electron microscopy. Analysis of DNA integrity by conventional gel electrophoresis failed to detect internucleosomal DNA cleavage. Resolution of DNA by field inversion gel electrophoresis showed fragments of 50 kilobases (kb). Etoposide induced the transient appearance of an additional DNA band of > 600 kb, which was temporally coincident with DNA-protein complex formation and was rapidly reversible upon drug removal. This DNA band was not observed after N-methylformamide treatment. In situ DNA end-labeling showed the incorporation of biotinylated dUTP into 50-kb DNA fragments but not etoposide-induced DNA fragments of > 600 kb. DNA end-labelling with terminal deoxynucleotidyltransferase was therefore not dependent upon intenucleosomal DNA cleavage, and fragments of approximately 50 kb were characterized by free 3'-OH termini that were not occluded by topoisomerase II protein. Although we considered that topoisomerase II potentially played an active role in the fragmentation of higher order chromatin during apoptosis, the results showed that DNA cleavage by topoisomerase II induced reversible, protein-associated fragments of > 600 kb and not irreversible cleavage to 50-kb fragments. The reversible cleavage of DNA to fragments of > 600 kb appears to be a signal for the engagement of apoptosis and is not an initial step in the sequential unwinding of chromatin.

Apoptosis↗