Drug toxicity in E. coli cells expressing human topoisomerase I.
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Agar↗
Biomedical subjects
S T Taylor
Publications and source records attributed to S T Taylor.
Epigenetic determinants of resistance to etoposide regulation of Bcl-X(L) and Bax by tumor microenvironmental factors.
BACKGROUND: Epigenetic factors (i.e., alterations of gene activity not involving mutations), as well as genetic changes in surviving cancer cells, may play an important role in drug resistance following cancer chemotherapy-a common cause of tumor relapse. Bcl-2 family proteins are central to the regulation of apoptotic cell death and modulate drug sensitivity. We investigated how survival signals in the cellular microenvironment affect the expression, protein conformation, and protein-protein interactions of the Bcl-2 family proteins Bax and Bcl-x(L) and how changes in response to microenvironmental signals alter the response of cancer cells to the drug etoposide. METHODS: JLP119 human B-lymphoma cells were treated with etoposide (40 microM) and then cultured in the presence of an activating anti-CD40 antibody, vascular cellular adhesion molecule-1 (VCAM-1)-to activate VLA-4 (alpha4beta1) integrin, and interleukin 4. Cell fate was monitored after etoposide treatment with or without these microenvironmental signals. Bcl-x(L) gene transcription and protein levels of Bcl-x(L) and Bax were measured by northern and western blotting, respectively. Nuclear translocation of transcription factor NF-kappaB was monitored by immunofluorescence and inhibited by (E)-capsaicin. Bax conformation and Bax-Bcl-x(L) interactions were monitored by immunofluorescence and immunoprecipitation, respectively. RESULTS: Microenvironmental survival signals produced statistically significant reductions in etoposide-induced apoptotic cell death, from 84.6% (95% confidence interval [CI] = 76.7%-92.4%) to 21.3% (95% CI = 19.5%-23.0%); P<.001. Activation of surface protein CD40 increased Bcl-x(L) protein levels via an (E)-capsaicin-inhibitable activation of NF-kappaB; i.e. , (E)-capsaicin restored etoposide sensitivity. Interleukin 4 had no effect on Bcl-x(L) protein levels but accelerated the increase in Bcl-x(L) protein associated with CD40 activation. VCAM-1- and interleukin 4-mediated signals diminished conformational changes in Bax protein and prevented the etoposide-induced disruption of constitutive Bax-Bcl-x(L) binding. CONCLUSIONS: Microenvironmental factors reduce the sensitivity of a B-cell lymphoma to etoposide in vitro by modulating the expression and functions of Bax and Bcl-x(L). This interaction may provide a paradigm for epigenetically induced drug resistance in other tumors.
Antineoplastic Agents, Phytogenic↗
Survival signals within the tumour microenvironment suppress drug-induced apoptosis: lessons learned from B lymphomas.
The suppression of apoptosis is one mechanism by which tumours become drug resitant. Extracellular signals from the germinal centre (GC) of secondary lymphoid tissue can rescue B cells from physiological- and chemotherapy-induced apoptosis. Such survival signals include CD40 receptor ligation, interleukin-4 (IL-4) receptor stimulation and the interaction of the integrin ligand VCAM-1 with its receptor. The GC environment was modelled in vitro by providing B lymphoma cells with these survival signals. JLP119 B lymphoma cells underwent apoptosis after exposure to the topisomerase II inhibitor etoposide and this was dramatically reduced when the cells were cultured in the GC system. CD40 receptor ligation resulted in increased levels of Bcl-XL. Etoposide diminished the binding between Bax and Bcl-XL but this was restored by IL-4 and VCAM-1 triggered signals. These data demonstrate combined effects of three microenvironmental signals on the Bcl-2 family and illustrate the potential importance of such signalling pathways in drug resistance of tumour cells.
Animals↗
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↗
The creation of a camptothecin-sensitive Escherichia coli based on the expression of the human topoisomerase I.
The synthesis of the human topoisomerase I (Topo I) in Escherichia coli (Ec) results in phenotypes consistent with the appearance of a DNA-relaxing activity. High-level expression was problematic and recloning the gene in a reduced-copy-number plasmid under more stringently regulated control produced a stable plasmid. An Ec strain with an inducible sensitivity to the eukaryotic Topo I poison, camptothecin (CPT), was constructed by introducing a mutation (imp4312) known to enhance the permeability of Ec to a wide variety of compounds. We are able to infer that CPT sensitivity in Ec involves DNA damage by noting elevated sensitivity in a repair-deficient recA strain and by observing the induction of a sulA::lac fusion following exposure to the drug.
Camptothecin↗
Evidence that chronic hyperprolactinemia effects skin temperature regulation through an opioid mechanism.
Studies were undertaken to evaluate the effects of chronic hyperprolactinemia (HYP) induced by the MtTW15 tumor on the thermoregulatory response of female rats to blockade of opiate receptors with naloxone. Both chronic administration of morphine and HYP cause a mild hyperthermia as evidenced by a 0.8-1.0 degrees C elevation in rectal temperature (Tr). Naloxone-precipitated morphine withdrawal caused a prompt increase (4.9 +/- 0.76 degrees C) in tail skin temperature (TST) and a subsequent decline in Tr (-2.8 degrees C). Similarly, naloxone administration to HYP rats caused a dramatic TST response which was coincident with the onset of severe HYP. This effect of naloxone was maximal at 7 weeks of tumor growth when a TST response of 4.8 +/- 0.3 degrees C was observed but was not evident prior to or 1 week following tumor inoculation, when serum prolactin levels were low. The TST response to naloxone in chronic HYP exhibited distinct pulses with an amplitude of 3.4 +/- 0.4 degrees C and a frequency of 2.2 +/- 0.5 pulses per 120 min. It appears that blockade of opiate receptors in HYP rats induces instability in the regulation of skin temperature as evident by recurrent episodes of TST surges. These effects of chronic HYP on the TST response to naloxone were not influenced by ovariectomy, suggesting that changes in ovarian secretions were not involved in the response. At 4 weeks of tumor growth, immunoreactive beta-endorphin concentrations in the medial basal hypothalamus, preoptic area-anterior hypothalamus and the neurointermediate lobe of the pituitary were decreased by 59, 28 and 47%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Rapid induction of IGF-IR signaling in normal and tumor tissue following intravenous injection of IGF-I in mice.
The detection of IGF-IR signaling in animal models has important implications for determining the role of this receptor in normal physiology and tumor growth. While many reports have correlated changes in plasma IGF-I levels in vivo with biological responses, few have shown that altered IGF-I levels can directly affect signaling within normal or tumor tissue. Here, we present new data that shows how the intravenous (IV) injection of IGF-I can be used to directly examine IGF signaling at the tissue level. Tail-vein IV injection of IGF-I into mice resulted in a rapid and dose-dependent activation of the IGF-I receptor and downstream phosphorylation of Akt and ERK1/2 in liver, kidney, and mammary gland. Similarly, IV IGF-I rapidly stimulated signaling in HT-29 colorectal and in MCF-7 breast cancer xenografts. This study shows how IV IGF injection can be used to examine the signaling mechanisms used by IGF-IR, in both normal mammary tissue and during tumor growth, and may provide a model for the characterization of IGF inhibitors.
Animals↗