Mary Lynch

hnRNP K binds a core polypyrimidine element in the eukaryotic translation initiation factor 4E (eIF4E) promoter, and its regulation of eIF4E contributes to neoplastic transformation

ABSTRACT Translation initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a key role in regulation of cellular proliferation. Its effects on the m7GpppN mRNA cap are critical because overexpression of eIF4E transforms cells, and eIF4E function is rate-limiting for G1 passage. Although we identified eIF4E as a c-Myc target, little else is known about its transcriptional regulation. Previously, we described an element at position −25 (TTACCCCCCCTT) that was critical for eIF4E promoter function. Here we report that this sequence (named 4EBE, for eIF4E basal element) functions as a basal promoter element that binds hnRNP K. The 4EBE is sufficient to replace TATA sequences in a heterologous reporter construct. Interactions between 4EBE and upstream activator sites are position, distance, and sequence dependent. Using DNA affinity chromatography, we identified hnRNP K as a 4EBE-binding protein. Chromatin immunoprecipitation, siRNA interference, and hnRNP K overexpression demonstrate that hnRNP K can regulate eIF4E mRNA. Moreover, hnRNP K increased translation initiation, increased cell division, and promoted neoplastic transformation in an eIF4E-dependent manner. hnRNP K binds the TATA-binding protein, explaining how the 4EBE might replace TATA in the eIF4E promoter. hnRNP K is an unusually diverse regulator of multiple steps in growth regulation because it also directly regulates c-myc transcription, mRNA export, splicing, and translation initiation.

Singlet Oxygen, but not Oxidizing Radicals, Induces Apoptosis in HL-60 Cells¶

Abstract Oxidizing species (OS), produced by photosensitization or derived from cytotoxic agents, activate apoptotic pathways. We investigated whether two different OS, formed at the same subcellular sites, have equivalent ability to initiate apoptosis in HL-60 cells. Our previous work showed that absorption of visible light by rose bengal (RB) produces singlet oxygen exclusively, whereas absorption of ultraviolet A produces RB-derived radicals in addition to singlet oxygen. Singlet oxygen, but not the RB-derived radicals, induced nuclear condensation and DNA fragmentation into nucleosome-size fragments in a dose dependent manner. In contrast, the RB-derived radicals caused greater lipid oxidation than singlet oxygen. These results indicate that different OS, produced at the same subcellular sites, do not have the same ability to induce apoptosis and that the ability of an OS to initiate lipid oxidation does not necessarily correlate with its ability to induce apoptosis.

Comparison of photosensitized plasma membrane damage caused by singlet oxygen and free radicals

The efficiency and selectivity of photosensitized damage to membrane functions may be influenced strongly by the identity of the initial reactive species formed by the photosensitizer. To test this possibility, a photosensitizer, rose bengal (RB), was used that resides in the plasma membrane and which generates singlet molecular oxygen (1O2*) upon excitation with visible light, and radicals plus 1O2* upon excitation with UV radiation. With this approach, 1O2* and radicals are formed at the same locations in the plasma membrane. The response of three plasma membrane functions, namely, proline transport, membrane potential, and membrane impermeability to charged dye molecules, was assessed. The efficiencies of the responses in the presence and absence of oxygen were compared per photon absorbed by RB at two wavelengths, 355 nm (UV excitation) and 532 nm (visible excitation). The efficiency of oxygen removal before irradiation was assessed by measuring the RB triplet lifetime. The three membrane functions were inhibited more efficiently at 355 nm than at 532 nm in the presence of oxygen indicating that the radicals are more effective at initiating damage to membrane components than 1O2*. The ratio of photosensitized effects at the two wavelengths in the presence of oxygen was the same for two membrane functions but not for the third suggesting that 1O2* and radicals initiate a common mechanistic pathway for damage to some membrane functions but not to others. Removing oxygen reduced the efficiency of 355 nm-induced photosensitization by factors of 1.4 to 7. The sensitivity of the three membrane functions to 1O2*-initiated damage varied over a factor of 50 whereas radical initiated damage only varied by a factor of 15. In summary, these results indicate that radicals and 1O2* formed at the same locations in the plasma membrane vary in their efficiency and specificity for membrane damage but may, in some cases, operate by a common secondary damage mechanism in the presence of oxygen.

The MTAP-CDKN2A Locus Confers Susceptibility to a Naturally Occurring Canine Cancer

Background: Advantages offered by canine population substructure, combined with clinical presentations similar to human disorders, makes the dog an attractive system for studies of cancer genetics. Cancers that have been difficult to study in human families or populations are of particular interest. Histiocytic sarcoma is a rare and poorly understood neoplasm in humans that occurs in 15% to 25% of Bernese Mountain Dogs (BMD). Methods: Genomic DNA was collected from affected and unaffected BMD in North America and Europe. Both independent and combined genome-wide association studies (GWAS) were used to identify cancer-associated loci. Fine mapping and sequencing narrowed the primary locus to a single gene region. Results: Both populations shared the same primary locus, which features a single haplotype spanning MTAP and part of CDKN2A and is present in 96% of affected BMD. The haplotype is within the region homologous to human chromosome 9p21, which has been implicated in several types of cancer. Conclusions: We present the first GWAS for histiocytic sarcoma in any species. The data identify an associated haplotype in the highly cited tumor suppressor locus near CDKN2A. These data show the power of studying distinctive malignancies in highly predisposed dog breeds. Impact: Here, we establish a naturally occurring model of cancer susceptibility due to CDKN2 dysregulation, thus providing insight about this cancer-associated, complex, and poorly understood genomic region. Cancer Epidemiol Biomarkers Prev; 21(7); 1019–27. ©2012 AACR.

A wavelength dependent mechanism for rose bengal-sensitized photoinhibition of red cell acetylcholinesterase

A 2-fold enhancement in the efficiency of rose bengal-photosensitized inhibition of red cell acetylcholinesterase activity was observed upon excitation of the dye in the ultraviolet (UV) (313 nm) compared to irradiation in the visible (514 or 550 nm). The measurements of efficiency of photosensitized enzyme inhibition were based on the effect produced when the same number of photons are absorbed by rose bengal (RB) at each wavelength. The mechanism for this unexpected enhancement of RB photosensitization upon UV excitation was investigated. The yield of singlet oxygen (O2(1 delta g], detected by time-resolved luminescence at 1270 nm, was independent of excitation wavelength for RB. Radicals were produced upon irradiation of RB at 313 nm but not at 514 nm as detected by bleaching of N,N-dimethylnitrosoaniline (RNO). Irradiation of RB at 313 nm but not at 514 nm appeared to cause homolytic cleavage of carbon-iodine bonds in the dye because iodine radicals, I, detected as I2 were produced with a quantum yield of 0.0041 +/- 0.0005 upon excitation in the UV. Photolysis of I2 in the presence of RNO caused bleaching of the RNO absorption at 440 nm, apparently resulting from reaction of I with RNO. Thus, the enhanced photosensitization upon UV excitation of RB is attributed to formation of I and/or RB. These results indicate that radicals, produced with low relative yield but having high reactivity compared to O2(1 delta g), can contribute to photosensitized enzyme inhibition and may represent an alternative mechanism for photodynamic therapy.

RELAXATION OF VASCULAR SMOOTH MUSCLE INDUCED BY LOW-POWER LASER RADIATION

The relaxation of rabbit aorta rings induced by low‐power laser radiation was investigated in vitro to determine the location of the chromophore(s) responsible for this response and evaluate possible mechanisms. An action spectrum for relaxation was measured on rabbit thoracic aorta rings precontracted with norepinephrine. The decrease in isometric tension was measured during exposure to laser light (351–625 nm) delivered via a fiber optic to a small spot on the adventitial surface. The shortest UV wavelength (351 nm) was 35‐fold more effective than 390 nm and 1700‐fold more effective than 460 nm. Ultraviolet wavelengths also produced greater maximum relaxation (0.40–0.45) than visible wavelengths (0.20–0.25), suggesting that photovasorelaxation involves more than one chromophore.

c-myc Repression of TSC2 Contributes to Control of Translation Initiation and Myc-Induced Transformation

The c-myc oncogene plays a key role in cellular growth control, and translation initiation factors are among the transcriptional targets of Myc. Here, we describe a defect in translation initiation control in myc-null cells due to alterations in the mammalian target of rapamycin (mTOR) pathway. Myc loss increased sensitivity to dominant inhibition of eukaryotic translation initiation factor 4E function. Polysomal profiles of myc(-/-) cells revealed decreased translation initiation rates, which were accompanied by decreased 40S/60S ribosomal subunit ratios. Because the 40S small ribosomal subunit contains the key regulatory ribosomal protein S6 (rpS6), we considered that myc loss might affect expression of components of the mTOR signaling pathway that regulate rpS6 function. Among mTOR signaling components, Myc directly affected transcription of tuberous sclerosis 2 (TSC2), as shown by quantitative mRNA analysis and by Myc binding to its promoter in chromatin immunoprecipitation assays. Importantly, Myc acted as a strong and direct repressor for TSC2 expression because its loss increased TSC2 mRNA in myc-null and in HL60 shRNA experiments, activation of a mycER construct in myc(-/-) cells suppressed TSC2 induction in a myc box II-dependent manner, and mycER activation recruited Myc to the TSC2 promoter. The biological significance of the effect of Myc on TSC2 expression was shown by markedly reduced TSC2 mRNA levels in myc-transformed cells, stimulation of S6 kinase activity in myc-null cells by TSC2 siRNA, and decreased Myc-induced soft agar colony formation following retroviral transduction of TSC2. Together, these findings show that regulation of TSC2 can contribute to the effects of Myc on cell proliferation and neoplastic growth.

Activation of protein kinase C is required for protection of cells against apoptosis induced by singlet oxygen

We evaluated the role of protein kinase C (PKC) in the regulation of apoptosis triggered by singlet oxygen. Activation of PKC by short‐term 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) treatment inhibited apoptosis, whereas inhibition of PKC with several inhibitors potentiated this process. The antiapoptotic effect of TPA was accompanied by phosphorylation of extracelluar signal‐regulated kinase 1/2 (ERK1/2). Pretreatment of cells with MEK inhibitor, PD98059, inhibited TPA‐induced phosphorylation of ERK1/2 and the cytoprotective ability of TPA. These results suggest that activation of PKC in HL‐60 cells confers protection against apoptosis induced by singlet oxygen and that ERK1/2 mediates antiapoptotic signaling of PKC.

Growth controls connect: Interactions between c-myc and the tuberous sclerosis complex-mTOR pathway

Among other signals, cell growth is particularly controlled by the target of rapamycin (TOR) pathway that includes the tuberous sclerosis complex genes (TSC1/2), and through transcriptional effects regulated by c-myc. Overexpression of Drosophila Myc and TSC1/2 cause opposing growth and proliferation defects. Despite this relationship, direct regulatory connections between Myc and the TSC have only recently been evaluated. Other than studies of p53 regulation, little consideration has been given to transcriptional regulation of the TSC genes. Here we review evidence that transcriptional controls are potentially important regulators of TSC2 expression, and that Myc is a direct repressor of its expression. Since tuberin loss de-represses Myc protein, the connection between these two growth regulators is positioned to act as a feed-forward loop that would amplify the oncogenic effects of decreased tuberin or increased Myc. Further experiments will be needed to clarify the mechanisms underlying this important connection, and evaluate its overall contribution to cancers caused by TSC loss or Myc gain.

AN EFFICIENT OXYGEN INDEPENDENT TWO-PHOTON PHOTOSENSITIZATION MECHANISM

A novel oxygen‐independent photosensitization mechanism from the upper triplet state (Tn) of rose bengal has been demonstrated by selectively populating Tn by sequential two‐color laser excitation. Products formed from Tn inhibit red blood cell acetylcholinesterase and decrease viability of P388D1 mouse macrophage monocyte cells as measured by trypan blue exclusion assay. Laser flash photolysis studies indicate that Tn reacts efficiently, as evidenced by permanent photobleaching of T1 absorption, with chemical yields approaching unit efficiency. This mechanism may have application for oxygen deficient photosensitization under high intensity, pulsed laser irradiation.