Apoptosis mechanisms induced by shiga toxin 1 in the myelogeneous leukemia cell line THP-1 : a dissertation /
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
College Station, Tex. :
Texas A&M University System Health Science Center,
2006.
|
| Subjects: |
| Abstract: | ABSTRACT: Shiga toxin 1 (Stx1) is a member of the Shiga toxin family, a group of structurally and functionally related cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and enterohemorrhagic E. coli. Patients infected with Shiga toxin-producing bacteria develop bloody diarrhea, and are at risk for the development of acute renal failure and neurological impairment. We previously showed that Stx1 induced : i) TNF-α expression, and ii) apoptosis in the myelogenous leukemia THP-1 cell line. The kinetics and extent of Stx1 induced TNF-α production and apoptosis were dependent upon the state of cellular differentiation. Based on the findings, we hypothesized that Stx1 may trigger different apoptotic signaling pathways in monocytic vs. macrophage-like cells. We used Annexin V/PI staining, TUNEL staining, and caspase activity assays to examine the mechanisms of apoptosis induction. We show that monocytic THP-1 cells do not express TNFR-1 and TNFR-2, and the addition of exogenous TNF-α did not induce apoptosis of the cells. Thus, Stx1 appears to induce monocyte apoptosis by a TNF-α-independent mechanism. However, macrophage-like THP-1 cells express TNFRs, but surprisingly, soluble TNF-α also failed to trigger apoptosis. Treatment of macrophage-like THP-1 cells with anti-TNFR-1 or anti-TNF-α antibodies did not inhibit Stx1 induced apoptosis. Activation of caspases-1, -2 -3, -6, -8 and -9 was detected in both cell types. In monocytic cells, caspase-1, and -2 inhibitors did not block, and caspase-6 and -9 inhibitors partially block apoptosis. Caspase-8 and -3 inhibitors almost completely inhibited monocyte apoptosis. In macrophage-like cells, all caspase inhibitors blocked apoptosis to some degree, although caspases-3 and -9 played major roles in apoptosis. Levels of pro-caspase cleavage and onset of apoptosis correlated with the kinetics of caspase activation in both cell types. Bid cleavage, mitochondrial membrane potential disruption and cytochrome c release into the cytosol were detected in both cell types in the presence of Stx1. Macrophage-like THP-1 cells were less susceptible to Stx1-induced apoptosis compared to monocytic cells. We found that NF-kappaB and JNK MAPKs were involved in the regulation of anti-apoptotic factors, while p38 MAPKs appeared to regulate pro-apoptosis factors in macrophage-like cells treated with Stx1. Finally, we demonstrated that the ribotoxic stress response is associated with apoptosis induction in both cell types. Taken together, these data suggest that Stx-1 triggers distinct apoptotic pathways in THP-1 cells in a cell maturation dependent manner. |
|---|---|
| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies The Texas A&M University System Health Science Center in partial fulfillment for the requirements for the degree of Doctor of Philosophy December 2006." Approved as to style and content by: Vernon L. Tesh, David N. McMurray, Jane Welsh, Rajesh Miranda, John M. Quarles. |
| Physical Description: | xi, 159 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 133-157). |