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Michael J McCabe

Publications and source records attributed to Michael J McCabe.

13 recordsLinked to original sources
PubMed2006

p53 suppression of arsenite-induced mitotic catastrophe is mediated by p21CIP1/WAF1.

Arsenic trioxide, an acute promyelocytic leukemia chemotherapeutic, may be an efficacious treatment for other cancers. Understanding the mechanism as well as genetic and molecular characteristics associated with sensitivity to arsenite-induced cell death is key to providing effective chemotherapeutic usage of arsenite. Arsenite sensitivity correlates with deficient p53 pathways in multiple cell lines. The role of p53 in preventing arsenite-induced mitotic arrest-associated apoptosis (MAAA), a form of mitotic catastrophe, was examined in TR9-7 cells, a model cell line with p53 exogenously regulated in a tetracycline-off expression system. Arsenite activated G1 and G2 cell cycle checkpoints independently of p53, but mitotic catastrophe occurred preferentially in p53- cells. Cyclin B/CDC2(CDK1) stabilization and caspase-3 activation persisted in arsenite-treated p53- cells consistent with MAAA/mitotic catastrophe. N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, completely abolished arsenite-induced MAAA/mitotic catastrophe and greatly increased the mitotic index. WEE1 and p21CIP1/WAF1 inhibit cyclin B/CDC2 by CDC2 tyrosine-15 phosphorylation and direct binding, respectively. CDC2-Y15-P was transiently elevated in arsenite-treated p53+ cells but persisted in p53- cells. Arsenite induced p53-S15-P and p21CIP1/WAF1 only in p53+ cells. P21CIP1/WAF1-siRNA-treated p53+ cells were similar to p53- cells in mitotic index and cell cycle protein levels. p53-inducible proteins GADD45alpha and 14-3-3sigma are capable of inhibiting cyclin B/CDC2 but did not play a p53-dependent role in mitotic escape in TR9-7 cells. The data indicate that p53 mediates cyclin B/CDC2 inactivation and mitotic release directly via p21CIP1/WAF1 induction.

Arsenites↗
PubMed2006

Exit from arsenite-induced mitotic arrest is p53 dependent.

BACKGROUND: Arsenic is both a human carcinogen and a chemotherapeutic agent, but the mechanism of neither arsenic-induced carcinogenesis nor tumor selective cytotoxicity is clear. Using a model cell line in which p53 expression is regulated exogenously in a tetracycline-off system (TR9-7 cells) , our laboratory has shown that arsenite disrupts mitosis and that p53-deficient cells [p53(-)], in contrast to p53-expressing cells [p53(+)], display greater sensitivity to arsenite-induced mitotic arrest and apoptosis. OBJECTIVE: Our goal was to examine the role p53 plays in protecting cells from arsenite-induced mitotic arrest. METHODS: p53(+) and p53(-) cells were synchronized in G2 phase using Hoechst 33342 and released from synchrony in the presence or absence of 5 microM sodium arsenite. RESULTS: Mitotic index analysis demonstrated that arsenite treatment delayed exit from G2 in p53(+) and p53(-) cells. Arsenite-treated p53(+) cells exited mitosis normally, whereas p53(-) cells exited mitosis with delayed kinetics. Microarray analysis performed on mRNAs of cells exposed to arsenite for 0 and 3 hr after release from G2 phase synchrony showed that arsenite induced inhibitor of DNA binding-1 (ID1) differentially in p53(+) and p53(-) cells. Immunoblotting confirmed that ID1 induction was more extensive and sustained in p53(+) cells. CONCLUSIONS: p53 promotes mitotic exit and leads to more extensive ID1 induction by arsenite. ID1 is a dominant negative inhibitor of transcription that represses cell cycle regulatory genes and is elevated in many tumors. ID1 may play a role in the survival of arsenite-treated p53(+) cells and contribute to arsenic carcinogenicity.

Apoptosis↗
PubMed2005

Inorganic mercury inhibits the activation of LAT in T-cell receptor-mediated signal transduction.

Little is known as to the molecular mechanisms involved with mercury intoxication at very low levels. Although the mechanism is not known, animal studies have nevertheless shown that low levels of mercury may target the immune system. Inorganic mercury (Hg2+) at very low (but non-toxic) levels can disrupt immune system homeostasis, in that genetically susceptible rodents develop idiosyncratic autoimmune disease, which is associated with defective T-cell function. T lymphocyte function is intimately coupled to the T-cell receptor. We have previously reported that on a molecular level, low concentrations of Hg2+ disrupt signaling from the T-cell receptor by interfering with activation of Ras and ERK MAP kinase. In this report we expand upon those results by showing that in T lymphocytes exposed to low concentration of Hg2+, Ras fails to become properly activated because upstream of Ras in the T cell signal transduction pathway, the important scaffolding element Linker for Activation of T Cells (LAT) fails to become properly phosphorylated. Hypo-phosphorylation of LAT occurs, because upstream of LAT, the LAT reactive tyrosine kinase ZAP-70 is also not properly activated in Hg2+ treated cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Lead enhances CD4+ T cell proliferation indirectly by targeting antigen presenting cells and modulating antigen-specific interactions.

Although Pb is a well-known immunotoxicant, its mechanism of action is not well understood. Low levels of Pb (approximately 1 microM) markedly enhance the proliferative T cell response in mixed lymphocyte culture (MLC), a process we have termed allo-enhancement. As Pb allo-enhancement occurs whether alloantigen presenting cells (APC) are derived from C57BL/6 or BALB.B10, the allo-reactive T cells involved are likely to be specific for peptide in the context of the IA(b) molecule as the IE molecule is null in H-2(b) mice. Analysis of T cell division in MLC with Pb treatment indicated that there was no significant difference between Pb and non-Pb-treated cultures until day 4 when the frequency of proliferating T cells was much greater than in non-treated cultures. Our data suggest that this increased proliferation is not coupled with increased IL-2 levels in the media as these were actually decreased with Pb treatment and that Pb-induced enhancement in the allo-proliferative response is only partially dependent upon IL-2. Pb allo-enhancement is abrogated when stimulating allo-APCs are paraformaldehyde-fixed, and T cell proliferation stimulated by concanavalin A is not enhanced with Pb treatment, suggesting that the APC is the proximate target of Pb in allo-MLC. Pb allo-enhancement does not occur when T cells respond to irradiated allo-B cells, alone; however, it is restored when syngeneic CD11c-enriched cells are added. Of the CD11c-enriched splenocytes, the fraction that is adherent after 24 h, consistent with macrophages, appears to be the cell type targeted by Pb. Using T cells from DO11.10 transgenic mice, we determined that the effect of Pb is centered around specific p:MHC interactions and that enhanced costimulation is an unlikely mechanism for Pb allo-enhancement.

Animals↗
PubMed2005

Arsenite delays progression through each cell cycle phase and induces apoptosis following G2/M arrest in U937 myeloid leukemia cells.

Arsenic is a well known toxicant and carcinogen that is also effective as a chemotherapeutic in the treatment of acute promyelocytic leukemia. Although its effects on humans are well documented, arsenic's mechanism of action is not well understood. Its ability to act as a carcinogen and as a chemotherapeutic seems paradoxical. However, cancer cell transformation and cancer cell destruction can both occur through perturbations of the cell cycle machinery, making cell cycle function a likely target of arsenic action. Arsenic has previously been shown to inhibit cancer cell cycle progression, but the targeted cell cycle phase has been debated. This study was designed to identify the cell cycle phase at which U937 cells are most sensitive to arsenite-induced growth inhibition. Centrifugal elutriation was used to divide asynchronous cell cultures into specific cell cycle phase-enriched fractions. These fractions were monitored for cell cycle phase progression in the presence and absence of sodium arsenite. We found an overall reduction in cell cycle progression rather than induction of arrest at one specific checkpoint. G(2)/M is the phase most sensitive to arsenite-induced apoptosis. However, arsenite profoundly affects U937 cell growth by increasing the length of time it takes cells to transit each phase of the cell cycle. Future study of cell cycle inhibition by arsenic should consider that the effect may not be mediated by the major cell cycle checkpoints. Arsenic's ability to inhibit growth in any cell cycle phase may increase its value as a chemotherapeutic used together with other, more phase-selective agents, such as camptothecin.

Apoptosis↗
PubMed2005

Attenuation of CD95-induced apoptosis by inorganic mercury: caspase-3 is not a direct target of low levels of Hg2+.

Exposure to environmental mercury may be a factor that contributes to idiosyncratic autoimmune disease. Studies have demonstrated that inorganic, ionic mercury (i.e., Hg2+) modulates several lymphocyte signal transduction pathways, which may be a mechanism whereby Hg2+ dysregulates the immune response. The CD95/Fas apoptotic signaling pathway, which is of critical importance in regulating peripheral tolerance, is disrupted by low and environmentally relevant concentrations of Hg2+. Activation of the cysteine protease caspase-3 is a critical component of both CD95-mediated and TNF-alpha-induced apoptosis. The present work demonstrates that Hg2+ selectively disrupts death receptor mediated caspase-3 activation, where CD95-mediated caspase-3 activation is impaired in Hg2+ treated cells; whereas TNF-alpha-induced caspase-3 activation is not. Using the fluorogenic caspase-3 substrate, Ac-DEVD-7-amino-4-methyl coumarin, to measure caspase-3 enzyme activity as well as Western blotting to track processing of the caspase-3 proenzyme, we have considered the potential direct and indirect effects of Hg2+ on caspase-3. At relatively high concentrations and in a cell-free system, Hg2+ is capable of targeting the active site cysteinyl of caspase-3 resulting in enzyme inhibition. However, at more environmentally relevant exposures, Hg2+ does not gain access in appreciable quantities to the intracellular compartment where caspase-3 resides. Collectively, these data establish that Hg2+ impairs CD95-mediated apoptosis by targeting a plasma membrane proximal signaling event. By measuring the cellular Hg2+ content following various exposure conditions, we have determined that a cellular Hg2+ burden of approximately 50 ng/10(6) cells is sufficient to impair CD95-mediated caspase-3 activation. The present study furthers an understanding of the mechanism whereby relatively low and non-cytotoxic concentrations of Hg2+ may disrupt peripheral tolerance leading to sustained autoimmune disease.

Apoptosis↗
PubMed2005

Inorganic mercury dissociates preassembled Fas/CD95 receptor oligomers in T lymphocytes.

Genetically susceptible rodents exposed to low burdens of inorganic mercury (Hg2+) develop autoimmune disease. Previous studies have shown that low, noncytotoxic levels of Hg2+ inhibit Fas-mediated apoptosis in T cells. These results suggest that inhibition of the Fas death receptor pathway potentially contributes to autoimmune disease after Hg2+ exposure, as a consequence of disruption of peripheral tolerance. The formation of active death inducing signaling complexes (DISC) following CD95/Fas receptor oligomerization is a primary step in the Fas-mediated apoptotic pathway. Other recent studies have shown that Hg2+ at concentrations that inhibit apoptosis also inhibit formation of active DISC, suggesting that inhibition of DISC is the mechanism responsible for Hg2+-mediated inhibition of apotosis. Preassociated Fas receptors have been implicated as key elements necessary for the production of functional DISC. We present evidence in this study showing that low and nontoxic concentrations of Hg2+ induce the dissociation of preassembled Fas receptor complexes in Jurkat T cells. Thus, this Hg2+-induced event should subsequently decrease the amount of preassembled Fas available for DISC formation, potentially resulting in the attenuation of Fas-mediated apoptosis in T lymphocytes.

Biopolymers↗
PubMed2005

Blood lead level and risk of asthma.

Asthma and lead poisoning are prevalent among urban children in the United States. Lead exposure may be associated with excessive production of immunoglobulin E, possibly increasing asthma risk and contributing to racial disparities. The objective of this study was to examine racial differences in the association of blood lead level (BLL) to risk of developing asthma. We established and followed a cohort prospectively to determine asthma onset, using patient encounters and drug claims obtained from hospital databases. Participants were managed care enrollees with BLL measured and documented at 1-3 years of age. We used multiple variable analysis techniques to determine the relationship of BLL to period prevalent and incident asthma. Of the 4,634 children screened for lead from 1995 through 1998, 69.5% were African American, 50.5% were male, and mean age was 1.2 years. Among African Americans, BLL > or = 5 and BLL > or = 10 microg/dL were not associated with asthma. The association of BLL > or = 5 microg/dL with asthma among Caucasians was slightly elevated, but not significant [adjusted hazard ratio (adjHR) = 1.4; 95% confidence interval (CI), 0.7-2.9; p = 0.40]. Despite the small number of Caucasians with high BLL, the adjHR increased to 2.7 (95% CI, 0.9-8.1; p = 0.09) when more stringent criteria for asthma were used. When compared with Caucasians with BLL < 5 microg/dL, African Americans were at a significantly increased risk of asthma regardless of BLL (adjHR = 1.4-3.0). We conclude that an effect of BLL on risk of asthma for African Americans was not observed. These results demonstrate the need for further exploration of the complex interrelationships between race, asthma phenotype, genetic susceptibilities, and socioenvironmental exposures, including lead.

Black or African American↗
PubMed2003

Inorganic mercury attenuates CD95-mediated apoptosis by interfering with formation of the death inducing signaling complex.

Inorganic mercury (Hg2+) modulates several lymphocyte signaling pathways and has been implicated as an environmental factor linked to autoimmune disease. From the standpoint that autoimmune diseases represent disorders of cell accumulation, in which dysregulated apoptosis may be one mechanism leading to the accumulation of autoreactive lymphocytes, we have been investigating the influences of Hg2+ on CD95-mediated apoptosis. We demonstrate here that low and noncytotoxic concentrations of Hg2+ impair CD95 agonist-induced apoptosis in representative Type-I and Type-II T cell lines. Hg2+ treatment blocks the CD95 agonist-induced activation of initiator and effector caspases as well as the association between CD95 and the signaling adaptor, FADD. CD95 multimerization does not appear to be affected by Hg2+. Thus, the Hg2+ sensitive step within the CD95 death pathway is localized to the level of the death inducing signaling complex (DISC). Disruption of proper DISC formation may be a biochemical mechanism whereby Hg2+ contributes to autoimmune disease.

Apoptosis↗
PubMed2002

Arsenite disrupts mitosis and induces apoptosis in SV40-transformed human skin fibroblasts.

Chronic ingestion of arsenite-contaminated drinking water causes skin, bladder, and liver cancer. The mechanism of arsenite-induced carcinogenesis is unknown. Arsenite is known to disrupt mitosis and to delay transit through M phase in normal diploid fibroblasts. SV40-transformed human fibroblasts were observed to be hypersensitive to the cytotoxic and cytostatic effects of NaAsO(2) compared with normal diploid fibroblasts in concentration-response experiments. Five to 20 microM NaAsO(2) induced cytostasis in cycling normal diploid fibroblasts but not overt lethality in quiescent normal diploid fibroblasts. High concentrations of arsenite were overtly lethal in both cycling and quiescent cells. The IC50 for cycling SV40-transformed fibroblasts was 3.8 and 4.8 microM for the SV40-transformed lines GM4429 and GM0637, respectively, whereas, in cycling normal diploid fibroblasts (GM0024), the IC50 was 24.7 microM. Microscopic examination of NaAsO(2)-treated SV40-transformed fibroblasts suggested a concentration-dependent accumulation of cells in mitosis undergoing apoptosis. Treatment of SV40-transformed fibroblasts with 0-10 microM NaAsO(2) caused a concentration-dependent inhibition of cell proliferation, accumulation of cells having G2/M DNA contents, and increases in the mitotic index. Phase microscopy, annexin V binding, and electron microscopy demonstrated that arrested mitotic cells underwent apoptosis. These results indicate that SV40-transformation sensitizes cells to arsenite-induced mitotic arrest and induction of apoptosis in the mitotic cells.

Apoptosis↗
PubMed2002

Protein kinase C does not mediate the inhibitory action of lead on vitamin D3-dependent production of osteocalcin in osteoblastic bone cells.

The level of osteocalcin in serum is lower in lead-intoxicated children than in their normal counterparts. To explain this clinical observation, we investigated the mechanism of action of lead on vitamin D3-dependent osteocalcin production. Lead (5-20 microM) blocked the stimulating effects of vitamin D3 on osteocalcin production in cultured rat osteosarcoma cells (ROS 17/2.8). It is often suggested that activation of protein kinase C (PKC) is a critical mediator of the toxic actions of lead. Treatment of ROS cells with Gö6976, an inhibitor of PKC alpha and beta isozymes, produced similar effects as lead on vitamin D3-dependent osteocalcin production, while activation of PKC by phorbol-12-myristate-13-acetate (TPA) did not reverse or mimic this effect of lead. Thus activation of PKC is not consistent with the actions of lead on vitamin D3-dependent osteocalcin production. Measurement of PKC enzyme activity showed that 10 microM lead treatment does not activate or inhibit the activity of PKC in ROS cells. Western blot analysis indicated that lead treatment does not translocate PKC alpha, beta, or zeta from cytosol to membrane. Therefore, we concluded that PKC does not mediate the cellular toxicity of lead on vitamin D3-dependent osteocalcin production.

Alkaline Phosphatase↗
PubMed2002

Immunomodulation by metals.

Occupational or environmental exposure to metals is believed to affect human health adversely. One mechanism whereby metals can alter health is through modulation of immune homeostasis. Imbalances in immune regulation by metals can lead to inadequate or excessive production of inflammatory cytokines. Alternatively, metals can lead to inappropriate activation of lymphoid subsets involved in acquired immunity to specific antigens. Some resultant pathologies may include chronic inflammatory processes and autoimmune diseases. Metals may change the response repertoire by direct and indirect means by influencing expression of new antigens, new peptides, and/or antigen presentation by modifying the antigen-presenting complex. The differences in metal-induced immune responses between humans and the mechanisms of metal immunomodulation are discussed.

Adjuvants, Immunologic↗