Robert J. Shmookler Reis

Remarkable longevity and stress resistance of nematode PI3K-null mutants.

The great majority of lifespan‐augmenting mutations were discovered in the nematode Caenorhabditis elegans. In particular, genetic disruption of insulin‐like signaling extends longevity 1.5‐ to 3‐fold in the nematode, and to lesser degrees in other taxa, including fruit flies and mice. C. elegans strains bearing homozygous nonsense mutations in the age‐1 gene, which encodes the class‐I phosphatidylinositol 3‐kinase catalytic subunit (PI3KCS), produce progeny that were thought to undergo obligatory developmental arrest. We now find that, after prolonged developmental times at 15–20 °C, they mature into extremely long‐lived adults with near‐normal feeding rates and motility. They survive to a median of 145–190 days at 20 °C, with nearly 10‐fold extension of both median and maximum adult lifespan relative to N2DRM, a long‐lived wild‐type stock into which the null mutant was outcrossed. PI3K‐null adults, although a little less thermotolerant, are considerably more resistant to oxidative and electrophilic stresses than worms bearing normal or less long‐lived alleles. Their unprecedented factorial gains in survival, under both normal and toxic environments, are attributed to elimination of residual and maternally contributed PI3KCS or its products, and consequent modification of kinase signaling cascades.

Chromosomal Mapping of Osteopenia-Associated Quantitative Trait Loci Using Closely Related Mouse Strains

Peak bone mineral density (BMD) is a highly heritable trait in humans and is currently the best predictor of skeletal fragility underlying osteoporosis. The SAMP6 mouse strain displays unusually low BMD at maturity, and age‐dependent osteopenia associated with defective osteoblastogenesis. To identify quantitative trait loci (QTLs) influencing bone density, we constructed crosses between SAMP6 and either AKR/J or SAMP6, two related mouse strains of higher peak BMD. Due to common ancestry of these strains, intercross parents differed at only 39–40% of 227 highly‐polymorphic genotyping markers, thus restricting our search to this informative portion of the genome and reducing the number of mice required for QTL significance. Using dual energy X‐ray absorptiometry (DEXA), we measured spinal BMD in F2 cross progeny at 4 months of age, and selectively genotyped those in the highest and lowest quartiles for BMD. Based on linear regression of bone density on genotype, including Composite Interval Mapping to enhance mapping precision while adjusting for effects of distal markers, we identified multiple QTLs significantly affecting spinal BMD; these were mapped to regions of chromosomes 2 (two sites, one confirmed in both crosses), 7, 11, 13 and 16. One of these loci had been previously identified as a significant bone‐density QTL, while 3 substantiate QTLs suggested by a low‐power study of 24 recombinant‐inbred mouse lines. Such recurrent appearance of QTLs, especially in crosses involving distantly‐related strains, implies that polymorphism at these loci may be favored by evolution and might underlie variation in peak bone density among humans.

Telomerase Inhibition and Cell Growth Arrest After Telomestatin Treatment in Multiple Myeloma

Purpose: The aim of this study was to test the efficacy of telomestatin, an intramolecular G-quadruplex intercalating drug with specificity for telomeric sequences, as a potential therapeutic agent for multiple myeloma. Experimental Design: We treated ARD, ARP, and MM1S myeloma cells with various concentrations of telomestatin for 7 days and evaluated for telomerase activity. Myeloma cells were treated with the minimal effective telomestatin concentration for 3–5 weeks. Every 7th day the fraction of live cells was determined by trypan blue exclusion, aliquots of cells were removed for various molecular assays, and the remaining cells were replated at the same cell number and at the same concentration of telomestatin. Telomere length, apoptosis, and gene expression changes were monitored as described in detail in “Materials and Methods.” Results: Telomestatin treatment led to inhibition of telomerase activity, reduction in telomere length, and apoptotic cell death in ARD, MM1S, and ARP myeloma cells. Gene expression profile after 1 and 7 days of telomestatin treatment revealed ≥2-fold change in only 6 (0.027%) and 51 (0.23%) of 33,000 genes surveyed, respectively. No changes were seen in expression of genes involved in cell cycle, apoptosis, DNA repair, or recombination. Conclusions: These results demonstrate that telomestatin exerts its antiproliferative and proapoptotic effects in myeloma cells via inhibition of telomerase and subsequent reduction in telomere length. We conclude that telomerase is an important potential therapeutic target for multiple myeloma therapy, and G-quadruplex interacting agents with specificity for binding to telomeric sequences can be important agents for additional evaluation.

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal

Caenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2‐2. CeGSTP2‐2 (the product of the gst‐10 gene) has the ability to conjugate the lipid peroxidation product 4‐hydroxynonenal (4‐HNE). Transgenic C. elegans strains were generated in which the 5′‐flanking region and promoter of gst‐10 were placed upstream of gst‐10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4‐4, an enzyme with particularly high catalytic efficiency for 4‐HNE. The localization of both transgenes was similar to that of native CeGSTP2‐2. The 4‐HNE‐conjugating activity in worm lysates increased in the order: control < mGsta4 transgenic < gst‐10 transgenic; and the amount of 4‐HNE‐protein adducts decreased in the same order, indicating that the transgenic enzymes were active and effective in limiting electrophilic damage by 4‐HNE. Stress resistance and lifespan were measured in transgenic animals (five independent lines each) and were compared with two independent control lines. Resistance to paraquat, heat shock, ultraviolet irradiation and hydrogen peroxide was greater in transgenic strains. Median lifespan of mGsta4 and gst‐10 transgenic strains vs. control strains was increased by 13% and 22%, respectively. In addition to the cause–effect relationship between GST expression and lifespan observed in the transgenic lines, correlative evidence was also obtained in a series of congenic lines of C. elegans in which lifespan paralleled the 4‐HNE‐conjugating activity in whole‐animal lysates. We conclude that electrophilic damage by 4‐HNE may contribute to organismal aging.

Dysfunctional homologous recombination mediates genomic instability and progression in myeloma

A prominent feature of most if not all cancers is a striking genetic instability, leading to ongoing accrual of mutational changes, some of which underlie tumor progression, including acquisition of invasiveness, drug resistance, and metastasis. Thus, the molecular basis for the generation of this genetic diversity in cancer cells has important implications in understanding cancer progression. Here we report that homologous recombination (HR) activity is elevated in multiple myeloma (MM) cells and leads to an increased rate of mutation and progressive accumulation of genetic variation over time. We demonstrate that the inhibition of HR activity in MM cells by small inhibitory RNA (siRNAs) targeting recombinase leads to significant reduction in the acquisition of new genetic changes in the genome and, conversely, the induction of HR activity leads to significant elevation in the number of new mutations over time and development of drug resistance in MM cells. These data identify dysregulated HR activity as a key mediator of DNA instability and progression of MM, with potential as a therapeutic target.

Reduced telomere length in ataxia-telangiectasia fibroblasts

Chromosomal instability with a high frequency of telomere fusion is characteristic of ataxia-telangiectasia cells both in vivo and in vitro. We have measured telomere length and found it to be consistently reduced in both diploid and SV40-transformed cells A-T fibroblasts, relative to control cells. We examined a few possible mechanisms which might account for telomeric length reduction, including telomerase activity in transformed cells and endogenous nuclease activities, but found no differences between A-T and control cells in these parameters.

Cancer in the Elderly: Basic Science and Clinical Aspects

The incidence of cancer increases progressively with age. Rearrangements of genomes have been found to accompany cellular aging. These factors, in concert with age-dependent alterations in immune function and host defense, may help to explain the increased risk of malignant disease in aged persons. The clinical presentation and natural history of neoplasia are also affected by aging. This conference reviews recent developments in these areas, examines the effects of drug use in the elderly and implications for management, and discusses current information on how age may influence the response of cancer to therapy.

Homologous recombination is elevated in some Werner-like syndromes but not during normal in vitro or in vivo senescence of mammalian cells

Werner syndrome (WS) is a recessive genetic condition associated with markedly reduced replicative lifespans of cells in culture, high chromosomal instability in vivo and in vitro, and premature appearance of many characteristics of normal aging, including an increased incidence of cancer. We have monitored plasmid homologous recombination frequencies in diploid fibroblasts from 6 Werner or Werner-like syndrome patients, following transfection with a plasmid substrate containing 2 overlapping fragments of the TN5 Neor gene. Plasmid DNA recovered from these cells was then assayed for homologous recombination by (a) transformation of recA- bacteria to Ampr (indicating total viable plasmid) or Neor (indicating viable recombinant plasmid), and (b) by limited-cycle polymerase chain reaction (PCR) to co-amplify a recombinant fragment containing the overlap region, and a control region of the same plasmid, without bacterial transformation. Bacterial assay data indicated that recombination rates in 3 of the 6 WS strains were significantly elevated above normal controls; 4 of 6 appeared elevated by PCR assay. The highest-recombination WS strain showed evidence of reduced degradation of transfected plasmid DNA. For this small sample of WS strains, clinical severity of WS was not well correlated with recombination rate as determined by either assay (Pearson r = 0.78, not significant, for PCR assay); elevated recombination may, however, define a subset of WS at greatest risk for cancer and/or atherosclerosis. PCR assay of a hyperoxia-resistant HeLa cell line, displaying substantially increased chromosome breakage, indicated increased recombination between direct-repeat fragments. Nevertheless, elevated recombination in WS strains is unlikely to be secondary to impaired replicative capacity characteristic of WS cells, or to defective repair of chromosome damage which is increased in WS, since recombination in non-WS strains was unaffected by passage level or repeated UV irradiation.

Cross-talk between LOX-1 and PCSK9 in vascular tissues

AIMS Lectin-like ox-LDL receptor-1 (LOX-1) plays an important role in inflammatory diseases, such as atherosclerosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates LDL receptor degradation and influences serum LDL levels. The present study was designed to investigate the possible interaction between PCSK9 and LOX-1. METHODS AND RESULTS In the first set of experiments, human vascular endothelial cells and smooth muscle cells were studied at baseline and after lipopolysaccharide (LPS) treatment (to create an inflammatory state). Both PCSK9 and LOX-1 were strongly induced by LPS treatment. To define the role of PCSK9 in LOX-1 expression, cells were transfected with siRNA against PCSK9, which resulted in reduced LOX-1 expression and function. On the other hand, cells exposed to recombinant hPCSK9 revealed enhanced LOX-1 expression (P < 0.05). To determine whether LOX-1 also regulates PCSK9, cultured cells in which LOX-1 was knocked down by siRNA expressed less PCSK9, whereas those transfected with hLOX-1 cDNA showed increased PCSK9 expression. The second set of experiments was carried out in wild-type (WT) and gene knockout (KO; LOX-1 and PCSK9) mice; LOX-1 KO mice showed much less PCSK9 (P < 0.05 vs. WT mice). PCSK9-KO mice showed much less LOX-1 (P < 0.05 vs. WT mice). Furthermore, we observed that mitochondrial reactive oxygen species (mtROS) plays an initiating role in the LOX-1/PCSK9 interaction, since mtROS induction enhanced and its inhibition reduced the expression of both PCSK9 and LOX-1. We also found that both LOX-1 and PCSK9 regulate adhesion molecules vascular cell adhesion molecule-1 expression. Finally, oxidized low-density lipoprotein and tumour necrosis factor-α, pro-inflammatory stimuli besides LPS, regulated PCSK9 expression that is mediated by the NF-κB signalling pathway. CONCLUSIONS These observations suggest that LOX-1 and PCSK9 positively influence each others expression, especially during an inflammatory reaction. mtROS appear to be important initiators of PCSK9/LOX-1 expression.

Lifespan extension in hypomorphic daf‐2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2‐2

Electrophilic stress caused by lipid peroxidation products such as 4‐hydroxynonenal (4‐HNE) and/or related compounds may contribute to aging. The major mode of 4‐HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2‐2, the product of the Caenorhabditis elegans gst‐10 gene, has the ability to conjugate 4‐HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2‐2 correlates highly with lifespan in a series of hypomorphic daf‐2 mutants of C. elegans. The overexpression of CeGSTP2‐2 in daf‐2 is abrogated in daf‐16; daf‐2 mutants, indicating that expression of the gst‐10 gene is modulated by insulin‐like growth factor signaling. To determine whether the relationship between CeGSTP2‐2 and lifespan is causal, we used RNAi to knock down CeGSTP2‐2. Treatment with gst‐10‐specific dsRNA decreased CeGSTP2‐2 protein in wild‐type N2 and in daf‐2 strains to an approximately equal level. The ability to conjugate 4‐HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf‐2 over N2 is due largely to the overexpression of CeGSTP2‐2. RNAi‐mediated knock‐down of CeGSTP2‐2 led to an increased susceptibility to 4‐HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf‐2 strains. These results indicate that CeGSTP2‐2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf‐2 mutants concordantly with other longevity assurance genes, probably via insulin‐like growth factor signaling.