Elena V. Kuligina

Regulatory Role of Small Nucleolar RNAs in Human Diseases

Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2′-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs.

Unbiased approach to profile the variety of small non-coding RNA of human blood plasma with massively parallel sequencing technology.

Objective: Understanding structures of circulating RNA expands fundamental knowledge of cell communications and signaling pathways as well as allows developing new molecular diagnostic approaches. The aim of this study was to deploy a new approach to sequencing cDNA library construction which expands the capabilities of high-throughput sequencing analysis of small non-coding RNAs. With the approach, we performed massively parallel sequencing of human blood plasma RNA to document profile of common and peculiar RNA species normally circulating in blood of healthy individuals. Methods: Total RNA was extracted from blood plasma samples of eight apparently healthy individuals. To obtain comprehensive cDNA libraries RNA was dephosphorylated and then 5′-phosphorylated. 5′-Phosphorylated total plasma RNA was ligated with adapters, reverse transcribed and eight personalized cDNA libraries were constructed. Libraries were sequenced with SOLiD™ technology. Results/conclusion: Fragments of rRNA, mitochondrial transcripts, microRNAs, fragments of scRNAs, snRNA and snoRNA, fragments of several mRNAs as well as the set of newly discovered transcripts were found to be permanent representatives of human blood plasma RNAs. Advanced mapping allowed to identify circulating herpes virus and enterobacterial transcripts. Documented profile of circulating RNA of healthy individuals provides basis for development of new approaches in research and diagnosis of human pathology.

Characterisation of circulating DNA by parallel tagged sequencing on the 454 platform

BACKGROUND Fragments of genomic DNA that can be isolated from the blood and body fluids of vertebrates are also known as circulating DNA. This DNA has widely been investigated as a biomarker for cancer and other diseases but the origin and significance of circulating DNA have not been elucidated to date. METHODS We used a parallel tagged sequencing method to sequence circulating DNA obtained from control individuals as well as cancer patients on the GSFLX sequencer (454 life sciences). RESULTS Circulating DNA sequenced on one 16th of a picotiter plate produced approximately 3600 unique circulating DNA sequences which were distributed over the human genome and a higher frequency of mutations was observed in cancer patients compared to healthy controls. CONCLUSION Circulating DNA represents genomic DNA in the blood of an individual, some sequence related differences might be evident between circulating DNA from cancer individuals and controls but distribution over the genome is similar.

Oligonucleosome DNA fragmentation of caspase 3 deficient MCF-7 cells in palmitate-induced apoptosis

Oligonucleosomal fragmentation of nuclear DNA is the late stage hallmark of the apoptotic process. In mammalian apoptotic cells fragmentation is catalyzed by DFF40/CAD DNase. DFF40/CAD primary activated through site‐specific proteolytic cleavage by caspase 3. The absence of caspase 3 in MCF‐7 leads to lack of oligonucleosomal DNA fragmentation under numerous apoptotic stimuli. In this study it was shown that palmitate induces apoptotic changes of nuclei and oligonucleosomal DNA fragmentation in casp3 deficient MCF‐7. Activation and accumulation of 40–50 kDa DFF40 like DNases in nuclei and cytoplasm of palmitate‐treated MCF‐7 were detected by SDS‐DNA‐PAGE assay. Microsomes of apoptotic MCF‐7 activate 40–50 kDa nucleases when incubated with human placental chromatin and induce oligonucleosomal fragmentation of chromatin in cell free system. Both DNases activation and chromatin fragmentation are suppressed in presence of caspase 3/7 inhibitor Ac‐DEVD‐CHO. Microsome associated caspase 7 is suggested to play the principal role in induction of oligonucleosomal DNA fragmentation of casp3 defitient MCF‐7.

Recombinant Analogs of a Novel Milk Pro-Apoptotic Peptide, Lactaptin, and Their Effect on Cultured Human Cells

We recently isolated and characterized a human milk peptide, lactaptin, which induced apoptosis of cultured human MCF-7 cells. Lactaptin was identified as a proteolytic fragment of human kappa-casein. Here, we generated two recombinant analogs of the peptide, RL1 and RL2, containing truncated and complete amino acid sequences of lactaptin, respectively. Analogs were produced in E.coli, purified and assayed for biological activity on cultured human MCF-7 cells. RL1 was shown to induce only a small decrease in cell viability, whereas RL2 lowered the viability of MCF-7 cells by 60%. This reduction in MCF-7 cell viability was associated with apoptosis, which was indicated by phosphatidilserine externalization and caspase-7 activation. The viability of A549 and Hep-2 cells was also reduced by RL2, albeit to a lesser degree than seen with MCF-7 cells; this reduced viability was not accompanied by apoptosis. Non-malignant human mesenchymal stem cells (MSC) were completely resistant to RL2 action.

Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin

PURPOSE Endometrial carcinoma is the most common gynecologic malignancy which is associated with a poor prognosis when diagnosed at an advanced stage; therefore, the discovery of efficacious new drugs is required to reinforce conventional chemotherapy. Short-term cultures of primary cells from endometrial tumors could be used for testing new anticancer therapeutics as well as for the development of personalized cancer therapy strategy. Here, the antitumor effect of a recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, was examined against primary human endometrial cancer cells. MATERIALS AND METHODS Primary cell cultures of malignant and normal human endometrium were performed by enzymatic digestion of endometrial tissue from biopsy material. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the messenger ribonucleic acid (mRNA) state of estrogen (ERs) and progesterone (PRs) hormone receptors and aromatase (Cyp 19) in cell cultures. Dynamic monitoring of cell adhesion and proliferation was made using the iCELLigence system (ASEA Biosciences). The sensitivity of cell cultures to conventional anticancer drugs and the lactaptin analog was estimated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and the iCELLligence system. RESULTS Established short-term primary cultures of endometrial cancer cells were ERα/ERβ/PR-positive and sensitive for RL2. The IC 50 values of doxorubicin and cisplatin were determined for all of the primary cultures designed. KE normal cells displaying low Cyp19 mRNA levels and high ERβ and PR mRNA levels were more resistant to RL2 treatment as well as to cisplatin and doxorubicin. CONCLUSIONS Our results indicate that the recombinant analog of lactaptin, RL2, exerts cytotoxic effects against primary hormone-dependent endometrial tumor cells in vitro with features of apoptosis.

A novel pro-apoptotic effector lactaptin inhibits tumor growth in mice models

Lactaptin, a human milk-derived protein, induces apoptosis in cultured tumor cells. We designed a recombinant analog of lactaptin (RL2) and tested its antitumor activity. The sensitivity of hepatocarcinoma A-1 (HA-1), Lewis lung carcinoma, and Ehrlich carcinoma to RL2 were tested to determine the most reliable in vitro animal model. HA-1 cells, which had the highest sensitivity to RL2, were transplanted into A/Sn mice to investigate RL2 antitumor activity in vivo. Investigation of the molecular effects of RL2 shows that RL2 induces apoptotic transformation of HA-1 cells in vitro: phosphatidylserine translocation from inner side of the lipid bilayer to the outer one and dissipation of the mitochondrial membrane potential. Repetitive injections of RL2 (5-50 mg/kg) for 3-5 days effectively inhibited ascites and solid tumor transplant growth when administered intravenously or intraperitoneally, without obvious side effects. The solid tumor inhibitory effect of RL2 (5 i.v. injections, cumulative dose 125 mg/kg) was comparable with that of cyclophosphamide at a therapeutic dose (5 i.v. injections, cumulative dose 150 mg/kg). In combination therapy with cyclophosphamide, RL2 had an additive antitumor effect for ascites-producing tumors. Histomorphometric analysis indicated a three-fold reduction of spontaneous metastases in the liver of RL2-treated mice with solid tumor transplants in comparison with control animals. Repeated RL2 treatment substantially prolonged the lifespan of mice with intravenously injected tumor cells. Recombinant analog of lactaptin effectively induced apoptosis of tumor cells in vitro and suppressed the growth of sensitive tumors and metastases in vivo.

Splicing by exon exclusion impaired by artificial box c/d RNA targeted to branch-point adenosine.

Box C/D small nucleolar RNAs guide the site‐specific 2′‐O‐ribose methylation of nucleotides in target rRNAs and snRNAs. In this study we used the ability of C/D box snoRNAs to guide modification of target RNAs with the aim of affecting the expression of predetermined human genes. We constructed an analogue of the human U24 box C/D RNA, the first antisense element of which was directed to induce 2′‐O‐ribose methylation of branch‐point adenosine in the intron of the human heat‐shock cognate protein (HSC8) pre‐mRNA. The second antisense element was directed to induce methylation of G1702 in human 18S rRNA. Constructed artificial box C/D RNA was obtained by in vitro transcription by T7 RNA polymerase from a synthetic DNA template. It was shown that passive transfection of human adenocarcinoma MCF‐7 cells with the artificial box C/D RNA, accompanied by heat shock, induced 2′‐O‐ribose methylation of targeted 18S rRNA guanosine G1702 and impaired splicing of the HSC8 pre‐mRNA. Derangement of HSC8 splicing was the result of exclusion of the exon downstream to the targeted intron branch point. The total efficacy of pre‐mRNA splicing impairment induced by artificial C/D box RNA was estimated as 6 to 10%.

Lactaptin Induces p53-Independent Cell Death Associated with Features of Apoptosis and Autophagy and Delays Growth of Breast Cancer Cells in Mouse Xenografts

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.

Artificial Box C/D RNAs Affect Pre-mRNA Maturation in Human Cells

Box C/D small nucleolar RNAs (snoRNAs) are known to guide the 2′-O-ribose methylation of nucleotides in eukaryotic ribosomal RNAs and small nuclear RNAs. Recently snoRNAs are predicted to regulate posttranscriptional modifications of pre-mRNA. To expand understanding of the role of snoRNAs in control of gene expression, in this study we tested the ability of artificial box C/D RNAs to affect the maturation of target pre-mRNA. We found that transfection of artificial box C/D snoRNA analogues directed to HSPA8 pre-mRNAs into human cells induced suppression of the target mRNA expression in a time- and dose-dependent manner. The artificial box C/D RNA directed to the branch point adenosine of the second intron, as well as the analogue directed to the last nucleotide of the second exon of the HSPA8 pre-mRNA caused the most prominent influence on the level of HSPA8 mRNAs. Neither box D nor the ability to direct 2′-O-methylation of nucleotides in target RNA was essential for the knockdown activity of artificial snoRNAs. Inasmuch as artificial box C/D RNAs decreased viability of transfected human cells, we propose that natural snoRNAs as well as their artificial analogues can influence the maturation of complementary pre-mRNA and can be effective regulators of vital cellular processes.