Mary Bakhanashvili

P53 in cytoplasm may enhance the accuracy of DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase

The tumor suppressor protein p53 displays 3′ → 5′ exonuclease activity and can provide a proofreading function for DNA polymerases. Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 is responsible for the conversion of the viral genomic ssRNA into the proviral DNA in the cytoplasm. The relatively low fidelity of HIV-1 RT was implicated as a dominant factor contributing to the genetic variability of the virus. The lack of intrinsic 3′ → 5′ exonuclease activity, the formation of 3′-mispaired DNA and the subsequent extension of this DNA were shown to be determinants for the low fidelity of HIV-1 RT. It was of interest to analyse whether the cytoplasmic proteins may affect the accuracy of DNA synthesis by RT. We investigated the fidelity of DNA synthesis by HIV-1 RT with and without exonucleolytic proofreading provided by cytoplasmic fraction of LCC2 cells expressing high level of wild-type functional p53. Two basic features related to fidelity of DNA synthesis were studied: the misinsertion and mispair extension. The misincorporation of noncomplementary deoxynucleotides into nascent DNA and subsequent mispair extension by HIV-1 RT were substantially decreased in the presence of cytoplasmic fraction of LCC2 cells with both RNA/DNA and DNA/DNA template-primers with the same target sequence. The mispair extension frequencies obtained with the HIV-1 RT in the presence of cytoplasmic fraction of LCC2 cells were significantly lower (about 2.8–15-fold) than those detected with the purified enzyme. In addition, the productive interaction between polymerization (by HIV-1 RT) and exonuclease (by p53 in cytoplasm) activities was observed; p53 preferentially hydrolyses mispaired 3′-termini, permitting subsequent extension of the correctly paired 3′-terminus by HIV-1 RT. The data suggest that p53 in cytoplasm may affect the accuracy of DNA replication and the mutation spectra of HIV-1 RT by acting as an external proofreader. Furthermore, the decrease in error-prone DNA synthesis with RT in the presence of external exonuclease, provided by cytoplasmic p53, may partially account for lower mutation rate of HIV-1 observed in vivo.

Oxidative stress causes telomere damage in Fanconi anaemia cells - a possible predisposition for malignant transformation.

Fanconi anaemia (FA) is an autosomal recessive and X‐linked disease characterized by severe genetic instability and increased incidence of cancer. One explanation for this instability may be the cellular hypersensitivity to oxidative stress leading to chromosomal breaks. This study explored the possible oxidative damage to telomeres of FA lymphocyte cell line, HSC536/N, and its possible effect on telomere function. We postulated that combination of oxidative damage with overexpression of telomerase may provide a possible model for malignant transformation in FA. The cells were grown in the presence of telomerase inhibitor and exposed for 1 month to H2O2 combined with various antioxidants. This exposure caused shortening of telomere length and damage to the telomere single stranded overhang, which was prevented by several oxidants. This shortening was associated with development of severe telomere dysfunction. Control cells did not exhibit this sensitivity to H2O2. Telomere dysfunction did not evoke damage response in FA cells, in contrast to normal P53 upregulation in control cells. Reconstitution of telomerase activity protected FA telomeres from further oxidative damage. These results suggest a scenario in which oxidative stress causes telomere shortening and ensuing telomere dysfunction may form the basis for malignant transformation in FA cells. Upregulation of telomerase activity in sporadic FA cells may perpetuate that process, thus explaining the malignant character of FA cells in vivo.

Effect of interferon on mouse cells chronically infected with murine leukaemia virus: kinetic studies on virus production and virus RNA synthesis.

NIH/3T3 cells chronically infected with the Moloney strain of murine leukaemia virus were incubated with interferon (IF). There was no effect on virus production during the first 4 h, but thereafter an antiviral state gradually developed, reaching a maximum at about 12 h. When IF was removed, the antiviral state (expressed in terms of inhibition of release of virus) remained constant for 10 h, after which there was an abrupt return to the normal rate of virus release. Analysis of IF-treated cells showed that there was a three to fourfold increase in the amount of virus RNA in the nucleus at 48 h after IF addition, and still a slight increase at 72 h. There were no increases in the amounts of virus RNA in the cytoplasm during 72 h after the addition of IF. These results agree with the postulate that IF inhibits a late stage in the maturation of virus in chronically infected cells.

Effect of imatinib on the signal transduction cascade regulating telomerase activity in K562 (BCR-ABL-positive) cells sensitive and resistant to imatinib.

OBJECTIVE Imatinib mesylate (IM) is a tyrosine kinase inhibitor selective for BCR-ABL and indicated for the treatment of chronic myeloid leukemia. It has recently been demonstrated that IM also targets other cellular components. Considering the significant role of telomerase in malignant transformation, we studied the effect of IM on telomerase activity (TA) and regulation in BCR-ABL-positive and -negative cells, sensitive and resistant to IM. MATERIALS AND METHODS Through combining telomeric repeat amplification protocol for detecting TA, reverse transcription polymerase chain reaction and Western blots for detecting RNA and protein levels of telomerase regulating proteins and fluorescence-activated cell sorting analysis, we showed that IM targets telomerase and the signal transduction cascade upstream of it. RESULTS IM significantly inhibited TA in BCR-ABL-positive and -negative cells and in chronic myeloid leukemia patients. TA inhibition was also observed in BCR-ABL positive cells resistant to IM at drug concentrations that did not lead to a reduction in BCR-ABL expression. In addition, a reduction in phosphorylated AKT and phosphorylated PDK-1 was also detected following IM incubation. CONCLUSIONS We demonstrate an inhibitory effect of IM on TA and on the AKT/PDK pathway. Because this effect was observed in cell expressing the BCR-ABL protein as well as cells not expressing it, and in cells sensitive as well as resistant to IM, it is reasonable to assume that the inhibitory effect of IM on TA is not mediated through known IM targets. The results of this study show that cells resistant to IM with regard to its effect on BCR-ABL could still be sensitive to IM treatment regarding other cellular components.

HTLV-1 Tax-induced NF-κB activation is synergistically enhanced by 12-O-tetradecanoylphorbol-13-acetate: mechanism and implications for Tax oncogenicity

Nuclear factor kappa B (NF-κB) factors regulate a wide range of physiological and oncogenic processes. Normally, these factors are transiently activated by specific external signals which induce their dissociation from inhibitors of κB (IκB) and subsequent translocation to the nucleus where p65 links to the cyclic adenosine monophosphate response element binding protein (CBP)–p300 and P/CAF coactivators that are essential for its transcriptional activity. The pathogenic potential of human T-cell leukemia virus type 1 (HTLV-1) Tax protein is partly ascribed to its capacity to constitutively activate NF-κB factors because constitutive activity of these factors play an important role in the pathophysiology of adult T-cell leukemia (ATL) and tropical spastic paraparesis–HTLV-1 associated myelopathy (TSP–HAM). In assessing the possibility of modulating Tax pathogenic potential by external factors, we focused here on 12-O-tetradecanoylphorbol-13-acetate (TPA) which is a potent protein kinase C (PKC) activator. There are conflicting reports regarding the effect of TPA and PKC on NF-κB. Therefore, we reassessed this issue and also investigated their influence on Tax-mediated activation of these factors. We found that TPA promoted NF-κB nuclear translocation and the DNA binding of p65 dimers through PKC activation. However, both TPA and ectopically expressed PKC had only a marginal effect on the transcriptional competence of these dimers, indicating that the DNA binding of such dimers is insufficient by itself for gene activation. Notably, however, both TPA and the ectopic PKC displayed strong synergistic enhancement of the Tax-induced activation of the NF-κB transcriptional function. In contrast, TPA and the ectopic PKC only slightly elevated the low activation of the NF-κB transcriptional capacity by cytoplasmic Tax mutants, indicating that the nuclear translocation of Tax was essential for this synergism. Subsequent experiments suggested that TPA contributed to this synergism by increasing the pool of free p65 which Tax could link to CBP and elevate, thereby, the amount of a p65–Tax–CBP ternary complex that could bind to NF-κB-responsive promoters and stimulate their expression. Finally, we demonstrated that this synergism operated also in HTLV-1-infected human T-cells. Earlier reports have shown a close linkage of pathological PKC-activating conditions (e.g., infectious and inflammatory diseases) to certain malignancies. On this ground, the present study suggests that such conditions may enhance the risk for ATL and TSP–HAM in HTLV-1 carriers by increasing the Tax-induced NF-κB activation.

Excision of Nucleoside Analogs from DNA by p53 Protein, a Potential Cellular Mechanism of Resistance to Inhibitors of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

ABSTRACT We investigated the ability of p53 in cytoplasm to excise nucleoside analogs (NAs). A decrease in incorporation of NAs by human immunodeficiency virus type 1 reverse transcriptase and their excision from DNA by p53, provided by the cytoplasmic fraction of LCC2 cells, suggest that p53 in cytoplasm may act as an external proofreader for NA incorporation.

Diagnosis of HIV-1 infection: Performance of Xpert Qual and Geenius supplemental assays in fourth generation ELISA-reactive samples

BACKGROUND Architect (AR) and Vidas (VD) fourth generation HIV screening immunoassays, which identify early stages of HIV infections, could have false positive results especially at low signal/cutoff (S/C) AR values. Geenius HIV1/2 (GS) is a specific confirmation line immunoassay that is not highly sensitive to early HIV infections. An HIV-1 RNA assay may better detect such infections. OBJECTIVES To evaluate all AR-VD reactive samples with GS results, and to assess Xpert Qual HIV-1 RNA assay (XQ) as an alternative to GS, in the first low S/C AR-VD-reactive samples from a tested individual. STUDY DESIGN First AR-VD-reactive-GS-tested results from all individuals with resolved HIV status, collected between March 2015 and March 2017 (n = 749), were retrospectively assessed. Samples with AR-VD-reactive-GS-discordant results and those with low S/C AR-VD-reactive results, were tested by XQ. Receiver operating characteristic (ROC) analysis of GS and XQ sensitivity/specificity was performed. RESULTS Overall, 94.1% (705/749) of AR-VD-reactive results were true HIV-1 positive. All samples with <3 S/C AR values were false positive. XQ resolved all first samples with AR-VD-reactive-GS-discordant results. The diagnostic accuracy of XQ in low (≤33 S/C) AR-VD-reactive samples was better than that of GS (97.6%, 81/83 versus 73.5%, 61/83, p < 0.01). ROC analysis for low S/C AR samples was optimal for pooled XQ and GS results. CONCLUSIONS Incorporating XQ in the current screening algorithm for the first AR-VD-reactive-GS-discordant samples may significantly reduce overall turn-around time of HIV-1 diagnosis.

The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway.

Abstract 4110: Downregulation of telomerase activity in response to epoxomicin in MM cells.

Background Proteasome inhibitors are effective drugs against multiple myeloma (MM). The mechanism of action of these pleiotropic agents has not been yet fully elucidated. The importance of telomerase activity (TA) in MM has been repeatedly demonstrated. Previously we showed that the first generation proteasome inhibitor bortezomib (B) inhibits TA in MM cells by both transcriptional and post-translational mechanisms and has a potential clinical significance. In the current study we evaluated the anti telomerase activity of the new generation of proteasome inhibitors, epoxomicin (EP) and MG132 (MG) in order to clarify whether telomerase inhibition represents a class effect. Methods MM cell lines, ARP-1, CAG, RPMI 8226 and U266 were exposed to EP or MG for 24-48 hours. Viability was assessed by the WST-1 and Trypan Blue exclusion assays, TA by the TRAP assay, hTERT expression by real time PCR, transcription factors (TF) binding by the ChIP assay and post-translational modifications were evaluated by IP and Western blot. Results EP and MG differentially downregulated the proliferation and TA in all MM cell lines. The downregulation of TA and hTERT (the gene encoding for telomerase) expression was faster in CAG than ARP-1 cells. EP was more potent than MG and therefore further mechanistic studies were performed on this compound. The inhibition of TA was mainly transcriptionally regulated and the phosphorylation of the enzyme was not changed. The binding of three positive regulator transcription factors (TF): SP1, c-Myc and NFκB to the hTERT promoter was decreased by EP in CAG cells as well as their total cellular concentrations. In ARP-1 cells the SP1 and c-MYC binding and content were similarly affected by EP while NFκB was not affected. Interestingly the transcription factor WT-1 (Wilms’ tumor-1) exhibited an increased binding to the hTERT promoter while its total cellular amount remained unchanged. Conclusions Our results demonstrate the effects of EP and MG on TA in MM. These results combined with our previous study of bortezomib define telomerase as a general target for proteasome inhibitors. The inhibitory effect on TA is exerted on several regulatory levels, transcriptional and post translational. SP1, C-Myc and NFkB were involved in mediating these effects. A novel finding of this study is the role of WT-1. WT-1 appears as a negative regulator of hTERT expression. The results of this study may contribute to future development of telomerase inhibition as a therapeutic modality in MM. Citation Format: Orit Uziel, Naama Shalem, Einat Beery, Jardena Nordenberg, Mary Bakhanashvili, Anna Gutkin, Meir Lahav. Downregulation of telomerase activity in response to epoxomicin in MM cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4110. doi:10.1158/1538-7445.AM2013-4110

Abstract 2997: Differential downregulation of telomerase activity by bortezomib in multiple myeloma cells: Regulatory pathways and clinical implications

Background: The proteasome inhibitor bortezomib (B) is an effective drug in multiple myeloma (MM). However, its exact mode of action is not yet fully understood. The importance of telomerase activity (TA) in the biology and prognosis MM is well established, but its response to B has not been assessed yet. In light of common signaling pathways connecting telomerase regulation and B known mechanisms of action, we surmised that the drug may affect the activity of telomerase in MM cells. Methods: Two MM cell lines, ARP-1 and CAG, were exposed to B for 24-72 hours. Viability was assessed by the WST-1 assay, TA by the TRAP assay, hTERT expression by real time PCR, transcription factors binding by the ChIP (Chromatin Immuno Precipitation) assay, post-translational modifications were evaluated by Western blot. Ex vivo studies included the isolation of mononuclear cells from bone marrow aspirates of MM patients scheduled to treatment with B. Results: B downregulated TA in all MM cell lines. However, the kinetics of this downregulation differed between the two lines. Whereas in ARP-1 cells TA decreased 24 hours after B treatment, in CAG cells the effect was observed only after 48 hours. These finding imply different regulatory mechanisms between these lines. In both lines B transcriptionally downregulated TA by inhibiting hTERT expression. ChIP analysis showed that SP-1 but not C-Myc or NFκB transcription factors mediate this transcriptional downregulation of telomerase. However, the two lines differed in phosphorylation of PKCα, which phosphorylates telomerase. Whereas in ARP-1 cells the phosphorylated form of PKCα was downregulated in response to B, the drug did not affect PKCα phosphorylation in CAG cells. These findings explain the different kinetics of telomerase downregulation and are in keeping with previous data showing different PKCα response between these two cell lines. We also show that telomerase phosphorylation decreased in response to B. These in vitro results were supported by ex vivo data from cells isolated from MM patients. Notably, the response of TA to B correlated with the clinical response of the patients to the drug. More over, this difference was dependent on the levels of p-PKCα in response to B. Conclusions: Taken together, our results shed light on the effects and mechanism of B on TA in MM. The differential response of the cells to B may depend on the response of pPKCα pathway to the drug. Telomerase inhibition by B may vary in subsets of MM patients in relation to their pPKCα dependency. A correlation between the effect of B on TA and resistance to B in MM was found. As such, the ex vivo assessment of TA in response to treatment may serve as a prognostic feature and add to the therapeutic armamentarium by the addition of telomerase inhibitors in a defined subsets of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2997. doi:10.1158/1538-7445.AM2011-2997