miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

SIRT3 protects from hypoxia and staurosporine-mediated cell death by maintaining mitochondrial membrane potential and intracellular pH.

Mitochondrial sirtuin 3 (SIRT3) mediates cellular resistance toward various forms of stress. Here, we show that in mammalian cells subjected to hypoxia and staurosporine treatment SIRT3 prevents loss of mitochondrial membrane potential (ΔΨ(mt)), intracellular acidification and reactive oxygen species accumulation. Our results indicate that: (i) SIRT3 inhibits mitochondrial permeability transition and loss of membrane potential by preventing HKII binding to the mitochondria, (ii) SIRT3 increases catalytic activity of the mitochondrial carbonic anhydrase VB, thereby preventing intracellular acidification, Bax activation and apoptotic cell death. In conclusion we propose that, in mammalian cells, SIRT3 has a central role in connecting changes in ΔΨ(mt), intracellular pH and mitochondrial-regulated apoptotic pathways.

Mitochondrial DNA variability modulates mRNA and intra-mitochondrial protein levels of HSP60 and HSP75: experimental evidence from cybrid lines.

To explore possible relationships between mitochondrial DNA (mtDNA) polymorphism and the expression levels of stress-responder nuclear genes we assembled five cybrid cell lines by repopulating 143B.TK(-) cells, depleted of their own mtDNA (Rho(0) cells), with foreign mitochondria with different mtDNA sequences (lines H, J, T, U, X). We evaluated, at both basal and under heat stress conditions, gene expression (mRNA) and intra-mitochondrial protein levels of HSP60 and HSP75, two key components in cellular stress response. At basal conditions, the levels of HSP60 and HSP75 mRNA were lower in one cybrid (H) than in the others (p = 0.005 and p = 0.001, respectively). Under stress conditions, the H line over-expressed both genes, so that the inter-cybrid difference was abolished. Moreover, the HSP60 intra-mitochondrial protein levels differed among the cybrid lines (p = 0.001), with levels higher in H than in the other cybrid lines. On the whole, our results provide further experimental evidence that mtDNA variability influences the cell response to stressful conditions by modulating components involved in this response. Sentence summary of the article: the results reported in the present study provide important experimental evidence that in human cells mtDNA variability is able to influence the cellular response to heat stress by modulating both the transcription of genes involved in this response and their intra-mitochondrial protein levels.

Characterization of a bidirectional promoter shared between two human genes related to aging: SIRT3 and PSMD13.

The human SIRT3 gene contains an intronic VNTR enhancer whose variability is correlated with life span. The SIRT3 5' flanking region encompasses the PSMD13 gene encoding the p40.5 regulator subunit of the 26S proteasome. Proteasome is a multicatalytic proteinase whose function declines with aging. SIRT3 and PSMD13 are linked in a head-to-head configuration (788-bp intergenic region). The molecular configuration of two genes that are both related to aging prompted us to search for shared regulatory mechanisms between them. Transfection experiments carried out in HeLa cells by deletion mutants of the PSMD13-SIRT3 intergenic region showed a complex pathway of coregulation acting in both directions. Furthermore, linkage disequilibrium (LD) analyses carried out in a sample of 710 subjects (18-108 years of age) screened for A21631G (marker of PSMD13), and for G477T and VNTR(intron5) (markers of SIRT3), revealed high LD, with significantly different PSMD13-SIRT3 haplotype pools between samples of centenarians and younger people.

Evidence that the mouse insulin receptor substrate-1 belongs to the gene family on which the promoter is activated by estrogen receptor alpha through its interaction with Sp1.

In the present study, the molecular mechanism underlying the up-regulatory effect of estradiol (E2) on mouse insulin receptor substrate-1 (IRS-1) promoter was investigated in CHO cells on which the same promoter had first been functionally characterized. The mouse IRS-1 promoter bears four consensus half Estrogen Responsive Elements (ERE) sequences and thirteen AP-1- and ten Sp1-binding elements. We performed molecular dissection of this promoter gene providing 3' different deleted constructs, containing the same AP-1 rich region with a progressively increased number of ERE half sites located downstream. None of these constructs was responsive to E2, while a downstream region (nt -1420 to -160) rich in GC elements was induced by E2. However, the latter region lost its intrinsic E2 responsiveness when the whole IRS-1 promoter was mutated for deletion in all four ERE half sites. Deletion analysis of the ERE half sites demonstrated that only ERE located at the position -1500 to -1495, close to the GC-rich region, was able to maintain the induced activatory effect of E2 on the IRS-1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the region containing the half ERE/Sp1 (nt -1500 to -1477) as the one conferring E2 responsiveness to the whole promoter. This effect occurs through the functional interaction between E2/ERalpha and Sp1.

Variability of the SIRT3 gene, human silent information regulator Sir2 homologue, and survivorship in the elderly.

The human sirtuin 3 (SIRT3) gene encodes a putative mitochondrial NAD-dependent deacetylase (SIRT3) which belongs to the evolutionary conserved family of sirtuin 2 proteins. Studies in model organisms have demonstrated that SIR2 genes control lifespan, while no data are available regarding a possible role of SIRT3 in human longevity. By analysing the genotype-specific survival function relevant to the G477T marker of SIRT3, we found that in males the TT genotype increases (p=0.0272), while the GT genotype decreases (p=0.0391) survival in the elderly. Since SIRT3 lies in a chromosomal region (11p15.5) where four genes potentially associated with longevity are located (HRAS1, Insulin-like Growth Factor 2, Proinsulin, and Tyrosine Hydroxylase) we tested for linkage-disequilibrium between G477T alleles and alleles of the above genes. The disequilibrium was not significant in any case, thus suggesting that SIRT3 itself, or a gene strictly linked to SIRT3, may have a role in human longevity.

The allele (A)(-110) in the promoter region of the HSP70-1 gene is unfavorable to longevity in women.

Heat shock proteins (HSPs) are crucial for maintenance of cell homeostasis and survival both during and after various stresses. The capability to cope with stress is believed to affect the chance of health and survival at organismal level. We have investigated whether the gene pool relevant to the (A/C)(-110) polymorphism in the promoter region of the HSP70-1 gene changes as the population ages and survival selection occurs. A total of 591 southern Italian subjects were enrolled in the study (263 males and 328 females; age range 18-109 years), free of clinically manifest diseases and with normal haemato-chemical parameters. A significant age-related decrease of the frequency of allele (A)(-110) was observed in females. The probability ratio of 0.403 (95% confidence interval [0.163, 0.910]) computed by considering female centenarians as cases and young women (18-49 years old) as controls showed that the (A)(-110) allele is unfavorable to longevity in females.

The influences on human longevity by HUMTHO1.STR polymorphism (Tyrosine Hydroxylase gene). A relative risk approach.

A new method based on the recently developed relative risk approach is introduced, and applied to data from Italian centenarian study (965 subjects aged from 13 to 109 years old) for investigating influences on longevity by Tyrosine Hydroxylase (TH) gene variability. The strategic parameterization enables the model to disentangle the various ways by which HUMTHO1.STR alleles (alleles 6, 7, 8, 9, 10*, 10, as defined according to the number of repeats) may contribute in reducing or increasing the hazard of death with different patterns of influences. Among all the alleles, we have found that allele 10* (10 imperfect repeats) shows a remarkable dominant and beneficial effect that reduces the log hazard of death in an additive manner. The results confirm that HUMTHO1.STR polymorphism is involved in the modulation of human longevity.

Triplet repeats, over-expanded in neuromuscular diseases, are under-represented in mammalian DNA: a survey of models.

Simple tandem repeats represent more than 1% of the human genome: occasionally they exhibit intergenerational expansibility and are associated with neuromuscular diseases. In transgenic mice the same sequences elicit similar symptoms, but do not expand. We have searched for di-, tri-, and tetra-repeats in the published DNA sequences of chromosomes 21 and 22 of Homo sapiens, as well as in more than five megabases of Mus musculus DNA. Human and murine DNA sequences show a shortage in frequency and base coverage of tri-repeats as compared to di- and tetra-repeats. In murine sequences the cumulative frequency of di-, tri-, and tetra-repeats and their overall base coverage are about threefold higher than in human. Models for both the shortage of tri-repeats found in man and mouse and for their dynamic expansions are discussed. We propose that some of the 10 possible tri-repeats may be more prone than others to assume unusual structures capable of interfering with DNA synthesis: hence the shortage of tri-repeats. If such repeats are located at the 3'end of a chain growing and thus approaching another chain annealed to the same template, as Okazaki fragments do during discontinuous and encumbered replication, a combination of strand displacement, template switch, and branch migration may produce structures resistant to removal, hence the expansion of tri-repeats.

A model for the involvement of Okazaki fragments maturation in the expansion of short tandem repeats.

We propose a model for the expansion of short tandem repeats (ESTR), a phenomenon which has been found to occur in human DNA and is associated with a dozen of neuromuscular diseases. The model is based mainly on theoretical considerations and recovers experimental data from the literature; it also finds support in preliminary results obtained by us in multiprimed polymerase chain reactions designed to assess the effects of a downstream primer on the fidelity of the elongation of an upstream one. The model links the occurrence of the ESTR to a defective maturation of the Okazaki fragments (OF), and in particular to an improper processing of their 3' termini. This may occur when the last OF approaches the 5' terminus of the previous one in a susceptible region of the template. It is postulated here that when a growing OF has progressed past the priming region and its main portion has been synthesized, upon approaching its conclusion, the final elongation may take place in a region of the template where certain triplets are repeated: in that case a series of aberrations on the elongation mechanism may occur. These aberrations could involve (a) the displacement of the 5' terminus of the penultimate, properly matured OF, enacted by the incoming 3' terminus of the last OF, (b) the switch of the latter to the displaced strand of the former as template, (c) the fold-back on itself of the growing 3' terminus of the last OF, (d) its assumption of an unusual structure because of the repetition, and (e) some impairment of its removal by structure-specific exo-endonuclease(s). Derangements of this last part of the process may trigger the ESTR.

Estradiol increases IRS-1 gene expression and insulin signaling in breast cancer cells.

This study demonstrates how the potentiating effects of E2 on insulin signaling in ER-positive breast cancer cells are consequent to an enhanced IRS-1 expression [corrected]. It induces an increase of both PI-3K/AKT and ERK1/2 activities. A direct action of E2 in the regulating mouse IRS-1 gene is also investigated in both Chinese hamster ovary and MCF-7 cells that are transfected with mouse IRS-1 regulatory sequences. The authors have reported, for the first time, how E2 induction of IRS-1 mRNA was correlated with a direct positive regulatory role of E2 on the IRS-1 promoter. This effect seems to be not strictly related to the cell type.

A procedure for cloning genomic DNA fragments with increasing thermoresistance.

Genomic DNAs have been cleaved by restriction or sonication, and the resulting double-stranded fragments have been exposed to increasing temperatures. This treatment may induce the helix-coil transition either in a single or in several steps, depending on the size and composition of the duplexes. Eventually, a critical temperature is reached at which each duplex melts completely and the two constitutive single strands separate. A transition interval can thus be defined for each duplex by the temperature at which the earliest strand separation takes place and that at which the most resistant double-stranded core collapses. If solutions containing a mixture of DNA duplexes are exposed to temperatures within their transition intervals, three kinds of molecules should originate: (1) duplexes that have not yet initiated the melting phase; (2) duplexes that have undergone only partial melting; and (3) single strands that derive from fully melted duplexes. If the heated solutions are quickly cooled to 0 degreesC, only the molecules from the first two classes can be ligated to a compatibly ended vector and cloned: class (1) are intact duplexes, and class (2) are molecules that snap immediately back to fully duplex structures: both are double-stranded. Conversely, the single strands of class (3) may not reanneal and thus be neither ligated nor cloned. We have tested the procedure on restricted coliphage lambda DNA, in view of its compartmentalized organization and known sequence. Then, we have applied it to human genomic DNA fragmented by sonication. After cloning of the available duplexes in a bacterial plasmid, libraries of molecules endowed with a progressively higher thermoresistance can be prepared for thermodynamic and genomic studies.

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.