Steven Buck

Selection for delayed senescence in Drosophila melanogaster

Understanding the mechanism whereby the aging process is controlled has proven to be a uniquely difficult biological problem. Many theories have been put forth offering explanations for the phenomenon of senescence on a variety of different levels ranging from cellular, biochemical, and physiological to genetic and evolutionary. Many of these explanations are nonexclusive, which adds redundancy to confusion in considering the whole body of theory. Many of the cellular and/or biochemical mechanisms proposed amount to little more than detailed discussions of various possible gene end-products, which are themselves the subject of genetic and evolutionary theories. And even among these, no single theory predominates. J. B. S. Haldane (1941) and P. B. Medawar (1952) advanced the first theory of senescence incorporating a modem genetic and evolutionary perspective on the aging process. Their theory postulates the existence of specialized age-of-onset modifier genes which repress the action of other genes that are deleterious until an advanced age has been reached. Little harm results from the expression of the mutations then, however, and senescence gradually ensues with their derepression. In this theory, selection modifies life span by simply increasing or decreasing the period over which such modifiers are effective. Williams (19 57) later expanded on this, introducing the notion that the genes influencing senescence might themselves act pleiotropically with reciprocal effects at early and late ages. In this theory, the beneficial effects of genes early in life are weighed in evolution against their late life effects; youthful vigor must be accompanied by an early senescence and short life, while a delayed senescence and long life occur at the cost of youthful vitality. Apart from further extension of these ideas by Hamilton (1966) and Emlen (1 970), no new major theories of the evolution of senescence have arisen since Williams (1957). One reason for this may be that until recently, the few experimental tests performed contributed comparatively little substantiating information toward these theories. Early attempts at modifying life span through artificial selection include that of Glass (1960), who withheld mating in Drosophila to enforce an early versus late age-specific pattern of reproduction. This produced a slight increase in the longevity of late-reproducing lines. Wattiaux (1968) also found an increase in longevity in Drosophila under selection for an agespecific pattern of reproduction. This was followed by Sokals (1970) study showing that continuous reproduction at an early age reduced median life span in Tribolium. Mertz (1975) found similar trends in an even later study. Taylor and Condra (1980) and Barclay and Gregory (1982) report changes in the longevity of Drosophila populations under rand K-selection or when exposed to predation. Concurrently with these, Lints and Hoste (1974, 1977) published the results of a well designed and extensive experiment that also selected for increased longevity in D. melanogaster through an early or late age-specific schedule of reproduction. But life span fluctuated wildly throughout the 13 generations of selection here, declining by 70% in the first few generations and then recovering. Further experiments (Lints et al., 1979)

Forward and reverse selection for longevity in Drosophila is characterized by alteration of antioxidant gene expression and oxidative damage patterns

Patterns of antioxidant gene expression and of oxidative damage were measured throughout the adult life span of a selected long-lived strain (La) of Drosophila melanogaster and compared to that of their normal-lived progenitor strain (Ra). Extended longevity in the La strain is correlated with enhanced antioxidant defense system gene expression, accumulation of CuZnSOD protein, and an increase in ADS enzyme activities. Extended longevity is strongly associated with a significantly increased resistance to oxidative stress. Reverse-selecting this long-lived strain for shortened longevity (RevLa strain) yields a significant decrease in longevity accompanied by reversion to normal levels of its antioxidant defense system gene expression patterns and antioxidant enzyme patterns. The significant effects of forward and reverse selection in these strains seem limited to the ADS enzymes; 11 other enzymes with primarily metabolic functions show no obvious effect of selection on their activity levels whereas six other enzymes postulated to play a role in flux control may actually be involved in NADPH reoxidation and thus support the enhanced activities of the ADS enzymes. Thus, alterations in the longevity of these Drosophila strains are directly correlated with corresponding alterations in; 1) the mRNA levels of certain antioxidant defense system genes; 2) the protein level of at least one antioxidant defense system gene; 3) the activity levels of the corresponding antioxidant defense system enzymes, and 4) the ability of the organism to resist the biological damage arising from oxidative stress.

Melatonin cytotoxicity in human leukemia cells: relation with its pro-oxidant effect

Melatonin has a variety of functions in human physiology and is involved in a number of pathological events including neoplastic processes. The tissue protective actions of melatonin are attributed to its antioxidant activity though, under certain conditions, melatonin might also exert oxidant effects, particularly in cancer cells. This study evaluated the effects of 10−5 and 10−3 m concentrations of melatonin on human leukemia cells. Moderate cytotoxic effects of melatonin at 10−3 m concentrations were observed in CMK, Jurkat and MOLT‐4 cells which was associated with significant reactive oxygen species (ROS) generation. Melatonin treatment was not associated with significant cytotoxicity in HL‐60 cells, although the generation of ROS was significantly increased. K562 and Daudi cells did not appear to be effected by melatonin treatment. Cellular membrane lipid peroxidation was not influenced by melatonin with the exception of CMK cells. Cell cycle kinetics were not affected in melatonin‐treated samples, again with the exception of CMK cells which showed increased apoptosis. Melatonin, therefore, induces the production of ROS that may be associated with cytotoxicity depending on the concentration of melatonin in some leukemia cells and does not appear to stimulate leukemia cell growth. These pro‐oxidant actions of melatonin may assist in limiting leukemic cell growth.

Comparison of DiOC6(3) uptake and annexin V labeling for quantification of apoptosis in leukemia cells and non-malignant T lymphocytes from children

Early during apoptosis, there is a reduction in mitochondrial transmembrane potential (MTP) and externalization of phosphatidylserine (PS) in cell membrane prior to eventual cell death. Flow cytometric detection techniques targeting these changes, reduction of DiOC(6)(3) uptake upon the collapse of MTP and annexin V binding to PS have been successfully used to detect apoptotic cells. These methods have given comparable results when cell lines were used. We compared the two different techniques, DiOC(6)(3) uptake and Annexin V-propidium iodide co-labeling in the quantification of cytarabine, vincristine and daunorubicin induced apoptosis on three leukemia cell lines (HL-60, CEM, U937), and bone marrow blasts from 26 children with acute myeloid leukemia, 14 with T cell acute lymphoblastic leukemia. Anti-Fas-induced apoptosis in culture-grown peripheral blood T lymphocytes on 18 samples from 9 children with non-malignant conditions were also studied by these techniques. Our results showed that there is a correlation (P < 0. 05) between the apoptosis rates measured by these two techniques for drug-induced apoptosis in myeloid and lymphoid blasts, and for anti-Fas mAb-induced apoptosis in T lymphocytes. This data suggests that reduction of the MTP and PS externalization may be common to many apoptotic pathways and techniques targeting either of these changes may be used in quantification of apoptosis in different clinical samples.

RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia.

RUNX1 (AML1) encodes the core binding factor alpha subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P < .001) lower than that in non-DS AMkL cases. Short hairpin RNA knockdown of RUNX1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivity to cytosine arabinoside, accompanied by significantly decreased expression of PIK3CD, which encodes the delta catalytic subunit of the survival kinase, phosphoinositide 3 (PI3)-kinase. Transcriptional regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease.

Synthesis and Discovery of High Affinity Folate Receptor-Specific Glycinamide Ribonucleotide Formyltransferase Inhibitors With Antitumor Activity

6-Substituted classical pyrrolo[2,3-d]pyrimidine antifolates with a three- to six-carbon bridge between the heterocycle and the benzoyl-L-glutamate (compounds 2-5, respectively) were synthesized starting from methyl 4-formylbenzoate and a Wittig reaction with the appropriate triphenylphosphonium bromide, followed by reduction and conversion to the alpha-bromomethylketones. Cyclocondensation of 2,4-diamino-4-oxopyrimidine with the alpha-bromoketones, coupling with diethyl-L-glutamate, and saponification afforded 2-5. Compounds 2-5 had negligible substrate activity for RFC but showed variably potent (nanomolar) and selective inhibitory activities toward Chinese hamster ovary cells that expressed FRalpha or FRbeta and toward FRalpha-expressing KB and IGROV1 human tumor cells. Inhibition of KB cell colony formation was also observed. Glycinamide ribonucleotide formyl transferase (GARFTase) was identified as the primary intracellular target of the pyrrolo[2,3-d]pyrimidines. The combined properties of selective FR targeting, lack of RFC transport, and GARFTase inhibition resulting in potent antitumor activity are unprecedented and warrant development of these analogues as antitumor agents.

Identical longevity phenotypes are characterized by different patterns of gene expression and oxidative damage.

Some years ago we applied simultaneously an identical regime of selection for late-life reproduction to several normal-lived sister lines (Ra and Rb) so as to produce several selected long-lived sister lines (La and Lb). The long-lived La and Lb sister lines had statistically identical longevity phenotypes and paraquat resistance phenotypes; however, we noticed some statistically different responses of the two strains at the biochemical level. Extensive work with the La strain showed that transcriptional alterations in antioxidant gene expression are robustly associated with its extended longevity. We decided to critically test the assumption of phenotypic equivalence by subjecting the Lb strain to the same series of molecular assays as was the La strain. The two sister strains are characterized by significantly different mechanisms and patterns of antioxidant gene expression, antioxidant enzyme activity, and oxidative damage. We find that the Lb strain appears to depend on the transcriptional activation of different genes than does the La strain, and on a post-translational up-regulation of at least one other antioxidant defense gene. The phenotypic equivalence observed at the organism level need not hold at the molecular genetic level. This finding suggests that there is more than one molecular mechanism by which antioxidant defense genes can bring about an increased resistance to oxidative stress. The theoretical and empirical implications of these findings are discussed.

MECHANISMS OF SYNERGISTIC ANTILEUKEMIC INTERACTIONS BETWEEN VALPROIC ACID AND CYTARABINE IN PEDIATRIC ACUTE MYELOID LEUKEMIA

Purpose: To determine the possibility of synergistic antileukemic activity and the underlying molecular mechanisms associated with cytarabine combined with valproic acid (VPA; a histone deacetylase inhibitor and a Food and Drug Administration–licensed drug for treating both children and adults with epilepsy) in pediatric acute myeloid leukemia (AML). Experimental Design: The type and extent of antileukemic interactions between cytarabine and VPA in clinically relevant pediatric AML cell lines and diagnostic blasts from children with AML were determined by MTT assays and standard isobologram analyses. The effects of cytarabine and VPA on apoptosis and cell cycle distributions were determined by flow cytometry analysis and caspase enzymatic assays. The effects of the two agents on DNA damage and Bcl-2 family proteins were determined by Western blotting. Results: We showed synergistic antileukemic activities between cytarabine and VPA in four pediatric AML cell lines and nine diagnostic AML blast samples. t(8;21) AML blasts were significantly more sensitive to VPA and showed far greater sensitivities to combined cytarabine and VPA than non-t(8;21) AML cases. Cytarabine and VPA cooperatively induced DNA double-strand breaks, reflected in induction of γH2AX and apoptosis, accompanied by activation of caspase-9 and caspase-3. Further, VPA induced Bim expression and short hairpin RNA knockdown of Bim resulted in significantly decreased apoptosis induced by cytarabine and by cytarabine plus VPA. Conclusions: Our results establish global synergistic antileukemic activity of combined VPA and cytarabine in pediatric AML and provide compelling evidence to support the use of VPA in the treatment of children with this deadly disease. Clin Cancer Res; 16(22); 5499–510. ©2010 AACR.

Genomic plasticity, energy allocations, and the extended longevity phenotypes of Drosophila

The antagonistic pleiotropy theory of the evolution of aging is shown to be too simple to fully apply to the situation in which Drosophila are selected directly for delayed female fecundity and indirectly for extended longevity. We re-evaluated our own previously reported selection experiments using previously unreported data, as well as new data from the literature. The facts that led to this re-evaluation were: (1) the recognition that there are at least three different extended longevity phenotypes; (2) the existence of metabolic and mitochondrial differences between normal- and long-lived organisms; and most importantly; (3) the observation that animals selected for extended longevity are both more fecund and longer-lived than their progenitor control animals. This latter observation appears to contradict the theory. A revised interpretation of the events underlying the selection process indicates that there is a two-step change in energy allocations leading to a complex phenotype. Initial selection first allows the up-regulation of the antioxidant defense system genes and a shift to the use of the pentose shunt. This is later followed by alterations in mitochondrial fatty acid composition and other changes necessary to reduce the leakage of H(2)O(2) from the mitochondria into the cytosol. The recaptured energy available from the latter step is diverted from somatic maintenance back into reproduction, resulting in animals that are both long-lived and fecund. Literature review suggests the involvement of mitochondrial and antioxidant changes are likely universal in the Type 1 extended longevity phenotype.

Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells.

Background Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML. Methodology Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis. Results Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis. Conclusion Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.