The effects of thymic hormones on the proliferation of human myelogenous leukemia cells

Thymosin fraction 5 (TF5), comprised of an array of small molecular weight peptides, partially purified from the adult bovine thymus gland, contains multiple biologically active humoral factors. TF5 influences T cell differentiation, lymphokine production and restores immune deficiencies in a variety of animal models in vitro and in vivo. Because TF5 generally enhances immune reactivity, we examined the effects of TF5 on the proliferation of HL-60 human promyelocytic leukemia cells. Direct viable dye cell counting, the MTT reaction, and the clonogenic potential of HL-60 cells were used to determine cell proliferation. The potency and efficacy of TF5 for the inhibition HL-60 cell proliferation was similar in all three measures of proliferation. In contrast to HL-60 cells, TF5 only induced a modest suppression of human melanoma CRL7686 cell proliferation. Known apoptosis inhibitors did not reverse the effect of TF5 on HL-60 cell proliferation indicating that TF5 acts through a non-apoptotic mechanism. The results of morphological analyses and the TUNEL assay demonstrate that TF5 does not induce apoptosis in HL-60 cells, suggesting that the mechanism of inhibition involves cytostasis. Further purification of TF5 by gel filtration chromatography produces a biologically active factor of approximately 20 kDa. Since the parent compound is composed of peptides less than 15 kDa, these results suggest the possible formation of oligomers. Electrophoresis of the active factor on polyacrylamide gels indicates that it contains at least four components in the 5--15 kDa molecular weight range; These results demonstrate that an activity in TF5 exerts an anti-proliferative effect in human myeloid leukemia cells through a cytostatic mechanism; these studies suggest that a thymic hormone immune surveillance mechanism may limit the onset of certain types of leukemia

Mitochondrial Genetic Background Modulates Bioenergetics and Susceptibility to Acute Cardiac Volume Overload

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial–nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the ‘mitochondrial paradigm’ for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress

Developmental exposure to second-hand smoke increases adult atherogenesis and alters mitochondrial DNA copy number and deletions in apoE(-/-) mice.

Cardiovascular disease is a major cause of morbidity and mortality in the United States. While many studies have focused upon the effects of adult second-hand smoke exposure on cardiovascular disease development, disease development occurs over decades and is likely influenced by childhood exposure. The impacts of in utero versus neonatal second-hand smoke exposure on adult atherosclerotic disease development are not known. The objective of the current study was to determine the effects of in utero versus neonatal exposure to a low dose (1 mg/m(3) total suspended particulate) of second-hand smoke on adult atherosclerotic lesion development using the apolipoprotein E null mouse model. Consequently, apolipoprotein E null mice were exposed to either filtered air or second-hand smoke: (i) in utero from gestation days 1-19, or (ii) from birth until 3 weeks of age (neonatal). Subsequently, all animals were exposed to filtered air and sacrificed at 12-14 weeks of age. Oil red-O staining of whole aortas, measures of mitochondrial damage, and oxidative stress were performed. Results show that both in utero and neonatal second-hand smoke exposure significantly increased adult atherogenesis in mice compared to filtered air controls. These changes were associated with changes in aconitase and mitochondrial superoxide dismutase activities consistent with increased oxidative stress in the aorta, changes in mitochondrial DNA copy number and deletion levels. These studies show that in utero or neonatal exposure to second-hand smoke significantly influences adult atherosclerotic lesion development and results in significant alterations to the mitochondrion and its genome that may contribute to atherogenesis

Experimental exposure regimen to second-hand smoke (SHS).

<p>apoE−/− mice on a C57BL/6J background were developmentally exposed to either filtered air or 1 mg/m³ total suspended particulate (TSP) SHS. The <i>in utero</i> group were exposed to 1 mg/m³ TSP from gestation days 1–19, followed by filtered air exposure until sacrifice at 12–14 weeks of age. The neonatal group were exposed to 1 mg/m³ TSP within 24 hours of birth until weaning at 3 weeks of age, followed by filtered air exposure until sacrifice at 12–14 weeks of age. Control mice were exposed exclusively to filtered air, until sacrifice at 12–14 weeks of age.</p

Oil red-O staining of aortas from developmental SHS exposed mice.

<p>Atherosclerotic lesion levels were assessed by quantifying the percent of oil red-O staining of whole aortas from mice exposed either to <i>in utero</i> or neonatal SHS (1 mg/m³ TSP). A) Bar graph demonstrating the percent levels of oil red-O positive staining aortic area relative to total aortic area from control, neonatal, and <i>in ute</i>ro exposed apoE −/− mice. An asterisk (*) indicates a significant difference exists compared to filtered air control (p≤0.001; n = 5, 6, and 8 for control, <i>in utero</i>, and neonatal groups, respectively). B) <i>En face</i> images of oil red-O stained aortas; arrows indicate presence of positively stained area.</p

Aconitase activity from aortas harvested from mice exposed to SHS during development.

<p>Aconitase is specifically inactivated by superoxide (O₂^.−) and peroxynitrate (ONOO⁻) and therefore provides an indirect measure of O₂^−. associated oxidant stress. A) Aconitase activity was measured in aorta homogenates from mice exposed to filtered air, neonatal SHS, or <i>in utero</i> SHS by following the production of cis-aconitate from isocitrate at 240 nm. An asterisk (*) indicates a significant difference exists compared to filtered air control (p≤0.001; n = 8/group). B) Immunoblot showing aconitase protein levels; the bar graph below depicts the quantification of relative aconitase protein levels to control (n = 8/group).</p

Pulmonary ozone exposure induces vascular dysfunction, mitochondrial damage, and atherogenesis

More than 100 million people in the United States live in areas that exceed current ozone air quality standards. In addition to its known pulmonary effects, environmental ozone exposures have been associated with increased hospital admissions related to cardiovascular events, but to date, no studies have elucidated the potential molecular mechanisms that may account for exposure-related vascular impacts. Because of the known pulmonary redox and immune biology stemming from ozone exposure, we hypothesized that ozone inhalation would initiate oxidant stress, mitochondrial damage, and dysfunction within the vasculature. Accordingly, these factors were quantified in mice consequent to a cyclic, intermittent pattern of ozone or filtered air control exposure. Ozone significantly modulated vascular tone regulation and increased oxidant stress and mitochondrial DNA damage (mtDNA), which was accompanied by significantly decreased vascular endothelial nitric oxide synthase protein and indices of nitric oxide production. To examine influences on atherosclerotic lesion formation, apoE−/− mice were exposed as above, and aortic plaques were quantified. Exposure resulted in significantly increased atherogenesis compared with filtered air controls. Vascular mitochondrial damage was additionally quantified in ozone- and filtered air-exposed infant macaque monkeys. These studies revealed that ozone increased vascular mtDNA damage in nonhuman primates in a fashion consistent with known atherosclerotic lesion susceptibility in humans. Consequently, inhaled ozone, in the absence of other environmental toxicants, promotes increased vascular dysfunction, oxidative stress, mitochondrial damage, and atherogenesis

Adult weights from apoE−/− exposed to developmental SHS.

<p>Bar graph depicting average weight of mice at 12–14 weeks of age, prior to sacrifice. An asterisk (*) indicates a significant difference (p<0.05) exists compared to control (n = 39, 30, and 19 for control, <i>in utero</i>, and neonatal groups, respectively).</p