Exploring the molecular biomarker utility of circCCT3 in multiple myeloma: A favorable prognostic indicator, particularly for R-ISS II patients.

Circular RNAs (circRNAs) are associated with the pathobiology of multiple myeloma (MM). Recent findings regarding circCCT3 support its involvement in the development and progression of MM, through microRNA sponging. Thus, we aimed to examine the expression of circCCT3 in smoldering and symptomatic MM and to assess its clinical importance. Three cell lines from plasma cell neoplasms were cultured and bone marrow aspirate (BMA) samples were collected from 145 patients with MM or smoldering MM. Next, CD138+ enrichment was performed in BMA samples, followed by total RNA extraction and reverse transcription. Preamplification of circCCT3 and GAPDH cDNA was performed. Finally, a sensitive assay for the relative quantification of circCCT3 using nested real-time quantitative polymerase chain reaction was developed, optimized, and implemented in the patients' samples and cell lines. MM patients exhibited significantly higher intracellular circCCT3 expression in their CD138+ plasma cells, compared to those from SMM patients. In addition, MM patients overexpressing circCCT3 had longer progression-free and overall survival intervals. The favorable prognostic significance of high circCCT3 expression in MM was independent of disease stage (either International Staging System [ISS] or revised ISS [R-ISS]) and age of MM patients. Interestingly, circCCT3 expression could serve as a surrogate molecular biomarker of prognosis in MM patients, especially those of R-ISS stage II. In conclusion, our study sheds new light on the significance of circCCT3 as a promising molecular marker for predicting MM patients' prognosis.

Small RNA-seq and clinical evaluation of tRNA-derived fragments in multiple myeloma: Loss of mitochondrial i-tRFHisGTG results in patients' poor treatment outcome.

Despite the substantial progress in multiple myeloma (MM) therapy nowadays, treatment resistance and disease relapse remain major clinical hindrances. Herein, we have investigated tRNA-derived fragment (tRF) profiles in MM and precursor stages (smoldering MM/sMM; monoclonal gammopathy of undetermined significance/MGUS), aiming to unveil potential MM-related tRFs in ameliorating MM prognosis and risk stratification. Small RNA-seq was performed to profile tRFs in bone marrow CD138+ plasma cells, revealing the significant deregulation of the mitochondrial internal tRFHisGTG (mt-i-tRFHisGTG) in MM versus sMM/MGUS. The screening cohort of the study consisted of 147 MM patients, and mt-i-tRFHisGTG levels were quantified by RT-qPCR. Disease progression was assessed as clinical end-point for survival analysis, while internal validation was performed by bootstrap and decision curve analyses. Screening cohort analysis highlighted the potent association of reduced mt-i-tRFHisGTG levels with patients' bone disease (p = 0.010), osteolysis (p = 0.023) and with significantly higher risk for short-term disease progression following first-line chemotherapy, independently of patients' clinical data (HR = 1.954; p = 0.036). Additionally, mt-i-tRFHisGTG-fitted multivariate models led to superior risk stratification of MM patients' treatment outcome and prognosis compared to disease-established markers. Notably, our study highlighted mt-i-tRFHisGTG loss as a powerful independent indicator of post-treatment progression of MM patients, leading to superior risk stratification of patients' treatment outcome.

Exploring the time-dependent regulatory potential of microRNAs in breast cancer cells treated with proteasome inhibitors.

PURPOSE: Breast cancer (BrCa) is a predominant type of cancer with a disparate molecular nature. MicroRNAs (miRNAs) have emerged as promising key players in the regulation of pathological processes in BrCa. Proteasome inhibitors (PIs) emerged as promising anticancer agents for several human malignancies, including BrCa, inhibiting the function of the proteasome. Aiming to shed light on the miRNA regulatory effect in BrCa after treatment with PIs, we used two PIs, namely bortezomib and carfilzomib. MATERIALS AND METHODS: Four BrCa cell lines of distinct molecular subtypes were treated with these PIs. Cell viability and IC50 concentrations were determined. Total RNA was extracted, polyadenylated, and reversely transcribed. Next, the levels of specific miRNAs with a significant role in BrCa were determined using relative quantification, and their regulatory effect was assessed. RESULTS: High heterogeneity was discovered in the levels of miRNAs in the four cell lines, after treatment. The miRNA levels fluctuate with distinct patterns, in 24, 48, or 72 hours. Interestingly, miR-1-3p, miR-421-3p, and miR-765-3p appear as key molecules, as they were found deregulated, in almost all combinations of cell lines and PIs. In the SK-BR-3 cell line, the majority of the miRNAs were significantly downregulated in treated compared to untreated cells, with miR-21-5p being the only one upregulated. Finally, various significant biological processes, molecular functions, and pathways were predicted to be affected. CONCLUSIONS: The diversity of pathways predicted to be affected by the diversity in miRNA expression after treatment with PIs paves the way for the recognition of new regulatory axes in BrCa.

miRNA-Based Technologies in Cancer Therapy.

The discovery of therapeutic miRNAs is one of the most exciting challenges for pharmaceutical companies. Since the first miRNA was discovered in 1993, our knowledge of miRNA biology has grown considerably. Many studies have demonstrated that miRNA expression is dysregulated in many diseases, making them appealing tools for novel therapeutic approaches. This review aims to discuss miRNA biogenesis and function, as well as highlight strategies for delivering miRNA agents, presenting viral, non-viral, and exosomic delivery as therapeutic approaches for different cancer types. We also consider the therapeutic role of microRNA-mediated drug repurposing in cancer therapy.

Discovery and Comprehensive Characterization of Novel Circular RNAs of the Apoptosis-Related BOK Gene in Human Ovarian and Prostate Cancer Cells, Using Nanopore Sequencing.

CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.

High Intratumoral i-tRF-GlyGCC Expression Predicts Short-Term Relapse and Poor Overall Survival of Colorectal Cancer Patients, Independent of the TNM Stage.

Colorectal cancer (CRC), one of the most prevalent types of cancer, requires the discovery of new tumor biomarkers for accurate patient prognosis. In this work, the prognostic value of the tRNA fragment i-tRF-GlyGCC in CRC was examined. Total RNA extraction from 211 CRC patient cancer tissue specimens and 83 adjacent normal tissues was conducted. Each RNA extract was subjected to in vitro polyadenylation and reverse transcription. A real-time quantitative PCR assay was used to quantify i-tRF-GlyGCC in all samples. Extensive biostatics analysis showed that i-tRF-GlyGCC levels in CRC tissues were significantly lower than in matched normal colorectal tissues. Additionally, the disease-free survival (DFS) and overall survival (OS) time intervals were considerably shorter in CRC patients with high i-tRF-GlyGCC expression. i-tRF-GlyGCC expression maintained its prognostic value independently of other established prognostic factors, as shown by the multivariate Cox regression analysis. Additionally, survival analysis after TNM stage stratification revealed that higher i-tRF-GlyGCC levels were linked to shorter DFS time intervals in patients with TNM stage II tumors, as well as an increased probability of having a worse OS for patients in TNM stage II. In conclusion, i-tRF-GlyGCC has the potential to be a useful molecular tissue biomarker in CRC, independent of other clinicopathological variables.

Novel circular RNAs of the apoptosis-related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing.

Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non-leukemic blood donors was extracted and reverse-transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first-strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non-leukemic blood donors. Lastly, a single-molecule resolution fluorescent in situ hybridization method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non-leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

MicroRNA-675-5p Overexpression Is an Independent Prognostic Molecular Biomarker of Short-Term Relapse and Poor Overall Survival in Colorectal Cancer.

Colorectal cancer (CRC) is the main cause of cancer-related deaths globally, highlighting the importance of accurate biomarkers for early detection and accurate prognosis. MicroRNAs (miRNAs) have emerged as effective cancer biomarkers. The aim of this study was to investigate the prognostic potential of miR-675-5p as a molecular prognostic biomarker in CRC. For this reason, a quantitative PCR assay was developed and applied to determine miR-675-5p expression in cDNAs from 218 primary CRC and 90 paired normal colorectal tissue samples. To assess the significance of miR-675-5p expression and its association with patient outcome, extensive biostatistical analysis was performed. miR-675-5p expression was found to be significantly downregulated in CRC tissue samples compared to that in adjacent normal colorectal tissues. Moreover, high miR-675-5p expression was associated with shorter disease-free (DFS) and overall survival (OS) in CRC patients, while it maintained its unfavorable prognostic value independently of other established prognostic factors. Furthermore, TNM stage stratification demonstrated that higher miR-675-5p levels were associated with shorter DFS and OS intervals, particularly in patients with CRC of TNM stage II or III. In conclusion, our findings suggest that miR-675-5p overexpression constitutes a promising molecular biomarker of unfavorable prognosis in CRC, independent of other established prognostic factors, including TNM staging.

miRNA-seq identification and clinical validation of CD138+ and circulating miR-25 in treatment response of multiple myeloma.

BACKGROUND: Despite significant advancements in multiple myeloma (MM) therapy, the highly heterogenous treatment response hinders reliable prognosis and tailored therapeutics. Herein, we have studied the clinical utility of miRNAs in ameliorating patients' management. METHODS: miRNA-seq was performed in bone marrow CD138+ plasma cells (PCs) of 24 MM and smoldering MM (sMM) patients to analyze miRNAs profile. CD138+ and circulating miR-25 levels were quantified using in house RT-qPCR assays in our screening MM/sMM cohort (CD138+ plasma cells n = 167; subcohort of MM peripheral plasma samples n = 69). Two external datasets (Kryukov et al. cohort n = 149; MMRF CoMMpass study n = 760) served as institutional-independent validation cohorts. Patients' mortality and disease progression were assessed as clinical endpoints. Internal validation was performed by bootstrap analysis. Clinical benefit was estimated by decision curve analysis. RESULTS: miRNA-seq highlighted miR-25 of CD138+ plasma cells to be upregulated in MM vs. sMM, R-ISS II/III vs. R-ISS I, and in progressed compared to progression-free patients. The analysis of our screening cohort highlighted that CD138+ miR-25 levels were correlated with short-term progression (HR = 2.729; p = 0.009) and poor survival (HR = 4.581; p = 0.004) of the patients; which was confirmed by Kryukov et al. cohort (HR = 1.878; p = 0.005) and MMRF CoMMpass study (HR = 1.414; p = 0.039) validation cohorts. Moreover, multivariate miR-25-fitted models contributed to superior risk-stratification and clinical benefit in MM prognostication. Finally, elevated miR-25 circulating levels were correlated with poor survival of MM patients (HR = 5.435; p = 0.021), serving as a potent non-invasive molecular prognostic tool. CONCLUSIONS: Our study identified miR-25 overexpression as a powerful independent predictor of poor treatment outcome and post-treatment progression, aiding towards modern non-invasive disease prognosis and personalized treatment decisions.

Human L-Dopa decarboxylase interaction with annexin V and expression during apoptosis.

l-Dopa Decarboxylase (DDC) is a pyridoxal requiring enzyme that catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (l-Dopa) to Dopamine (DA). The function of DDC in physiological and pathological biochemical pathways remains poorly understood, while the function and regulation of human DDC isoforms is almost completely elusive. We have shown that Annexin V, a fundamental apoptosis marker, is an inhibitor of l-Dopa decarboxylase activity. Here we show the interaction of both the full-length DDC and the truncated isoform alternative DDC (Alt-DDC) with Annexin V in human tissue and cell lines. Interestingly, DDC isoform expression is enhanced or remains unaffected following staurosporine (STS) treatment, despite increased levels of cytotoxicity and apoptosis. The findings presented here provide novel insights concerning the involvement of DDC in programmed cell death.

Circulating miR-146a and miR-134 in predicting drug-resistant epilepsy in patients with focal impaired awareness seizures.

OBJECTIVE: Epilepsy is one of the most prevalent neurologic disorders, causing serious psychological problems and reducing quality of life. Although 20 different antiepileptic drugs (AEDs) have been approved by the US Food and Drug Administration (FDA), 30% of patients have drug-resistant epilepsy (DRE). Considering the role of miR-146a and miR-134 in neuroinflammation and dendritic functionality, respectively, the aim of this study was the clinical evaluation of circulating miR-146a and miR-134 as novel noninvasive molecular markers for the prognosis of refractory epilepsy. METHODS: The study included 162 patients with focal impaired awareness seizures. Total RNA was extracted from serum samples spiked with synthetic cel-miR-39-3p for normalization purposes. First-strand complementary DNA (cDNA) synthesis was performed using microRNA-specific stem-loop primers, and hsa-miR-134/146a levels were quantified by quantitative polymerase chain reaction (qPCR). DRE was used as clinical end point event. Internal validation was performed by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit on disease prognosis. RESULTS: The circulating levels of both miR-134 and miR-146a were elevated in patients with drug-resistant seizures. The receiver-operating characteristic (ROC) curve and logistic regression analysis demonstrated that patients with increased circulating miR-134/146a levels are at significantly higher risk for developing DRE, independently of temporal lobe sclerosis, epilepsy duration, familial history, age at first seizure, age, body mass index (BMI), smoking behavior, and gender. Finally, decision curve analysis highlighted that the evaluation of circulating miR-134/146a led to superior clinical benefit for DRE prognosis and patients' risk stratification. SIGNIFICANCE: Elevated serum miR-134/146a levels are associated with a higher risk for AED-resistant epilepsy and could constitute novel noninvasive molecular markers to improve disease early prognosis and support precision medicine.

Identification of novel alternative transcripts of the human Ribonuclease κ (RNASEK) gene using 3' RACE and high-throughput sequencing approaches.

The human RNASEK gene encodes Ribonuclease κ, an endoribonuclease that belongs to a highly conserved protein family of metazoans. Recent evidence suggests that the mRNA levels of the RNASEK gene possess biomarker attributes in patients with prostate cancer. In the present study, we used 3' RACE and next-generation sequencing (NGS) to detect and identify novel RNASEK transcripts. Computational analysis of the NGS data revealed new alternative splicing events that support the existence of novel RNASEK alternative transcripts. As a result, eight RNASEK splice variants were discovered and their expression profile was analyzed with the use of nested PCR in a wide panel of human cell lines, originating from several cancerous and/or normal human tissues. Based on in silico analysis, six of the eight novel RNASEK transcripts are predicted to encode new protein isoforms, while the remaining two splice variants could be considered as nonsense-mediated mRNA decay (NMD) candidates.

Effect of Vinca Alkaloids on the Expression Levels of microRNAs Targeting Apoptosis-related Genes in Breast Cancer Cell Lines.

BACKGROUND: Treatment of cancer with natural chemotherapeutic agents induces apoptosis along with remarkable alterations in the expression of apoptosis-related genes. Deregulation of microRNA (miRNA) expression is implicated in several human malignancies. Vinca alkaloids compose a class of antimitotic drugs preventing cancer cells from dividing, leading to apoptosis. They are commonly used in clinical practice for breast cancer treatment. OBJECTIVE: The present study focused on the effects of vinca alkaloids (vincristine, vinblastine, and vinorelbine) on miRNA expression of treated breast cancer cells. METHODS: We investigated the effect of vincristine, vinblastine, and vinorelbine on the expression of oncogenic and tumor-suppressive miRNAs (miR-15a-5p, miR-16-5p, miR-21-5p, miR-25-3p, miR- 29b-3p, miR-125b-5p, miR-148a-3p, miR-214-3p, miR-221-3p, miR-222-3p, and miR-421), as well as on the expression of the apoptosis-related genes BAX, BCL2, TP53, and CDKN1B in BT-20 and SKBR- 3 breast adenocarcinoma cells. RESULTS: Treatment of BT-20 cells with vincristine, vinblastine, and/or vinorelbine resulted in upregulation of TP53 expression. However, no alterations in the mRNA levels of the pivotal BCL2 family members BAX and BCL2 were observed. On the other hand, treatment of SK-BR-3 cells with any of these vinca alkaloids led to an increase in the BAX/BCL2 mRNA ratio, implying the activation of the intrinsic apoptotic pathway. No concomitant alteration in TP53 expression was observed in treated SKBR- 3 cells. Regarding the miRNAs examined in this study, miR-222-3p expression exhibited the most remarkable modulations in both treated cell lines. CONCLUSION: This study suggests the possible involvement of miR-222-3p expression in breast cancer cell apoptosis, triggered by vincristine, vinblastine, and vinorelbine.

Loss of GAS5 tumour suppressor lncRNA: an independent molecular cancer biomarker for short-term relapse and progression in bladder cancer patients.

BACKGROUND: Bladder cancer (BlCa) heterogeneity and the lack of personalised prognosis lead to patients' highly variable treatment outcomes. Here, we have analysed the utility of the GAS5 tumour-suppressor lncRNA in improving BlCa prognosis. METHODS: GAS5 was quantified in a screening cohort of 176 patients. Hedegaard et al. (2016) (n = 476) and TCGA provisional (n = 413) were used as validation cohorts. Survival analysis was performed using recurrence and progression for NMIBC, or death for MIBC. Internal validation was performed by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit on disease prognosis. RESULTS: GAS5 levels were significantly downregulated in BlCa and associated with invasive high-grade tumours, and high EORTC-risk NMIBC patients. GAS5 loss was strongly and independently correlated with higher risk for NMIBC early relapse (HR = 2.680, p = 0.011) and progression (HR = 6.362, p = 0.035). Hedegaard et al. and TCGA validation cohorts' analysis clearly confirmed the association of GAS5 loss with NMIBC worse prognosis. Finally, multivariate models incorporating GAS5 with disease established markers resulted in higher clinical benefit for NMIBC prognosis. CONCLUSIONS: GAS5 loss is associated with adverse outcome of NMIBC and results in improved positive prediction of NMIBC patients at higher risk for short-term relapse and progression, supporting personalised prognosis and treatment decisions.

BCL2L12: a multiply spliced gene with independent prognostic significance in breast cancer.

Background Alternative splicing is a key process in carcinogenesis and, from a clinical aspect, holds great promises, as alternatively spliced variants have emerged as an untapped source of diagnostic and prognostic markers. Our aim was to assess the prognostic value of three recently recognized splice variants of the apoptosis-related gene, BCL2L12, in breast cancer (BC). Methods Total RNA was extracted from breast samples (150 BC and 80 tumor-adjacent normal tissues) and, following cDNA synthesis, a variant-specific qPCR was performed for the expressional quantification of BCL2L12 v.1, v.2 and v.4 transcript variants. Extensive statistical analysis, including bootstrap resampling and internal validation, was conducted in order to evaluate the associations of v.1, v.2 and v.4 expression with patients' clinopathological and survival data. Results All examined BCL2L12 variants were significantly upregulated in BC specimens compared to their non-cancerous counterpart (v.1, p<0.001; v.2, p=0.009; v.4, p=0.004). Increased BCL2L12 v.4 mRNA expression was associated with markers of unfavorable prognosis namely, advanced tumor grade (p=0.002), ER- (p=0.015)/PR- (p<0.001) negativity, Ki-67-positivity (p=0.007) and high NPI (Nottingham prognostic index) score (p=0.033). Moreover, v.4 was significantly overexpressed in women with triple negative BC (TNBC) and HER2-positive tumors compared to those harboring luminal tumors (p<0.001). Survival analysis disclosed that BCL2L12 v.2 overexpression, as a continuous variable ([HR]=0.45, 95% CI=0.17-0.82, p=0.010), is a strong and independent marker of favorable prognosis for BC patients. Interestingly, v.2 retains its prognostic value in patients with Grade II/III ([HR]=0.21, 95% CI=0.05-0.57, p=0.006) or HER2-positive/TNBC tumors ([HR]=0.25, 95% CI=0.05-0.74, p=0.042). Conclusions BCL2L12 v.1, v.2, v.4 are aberrantly expressed in BC. Their expressional analysis by cost-effective molecular methods could provide a novel molecular tool for BC management.

Expressional profiling and clinical relevance of RNase κ in prostate cancer: a novel indicator of favorable progression-free survival.

PURPOSE: Considering the unmet need for novel molecular tumor markers capable of improving prostate cancer (CaP) patients' management along with the fruitful results regarding the future use of ribonucleases (RNases) as molecular diagnostic and prognostic markers in CaP, we aimed to study the expressional profile of RNase κ in CaP and BPH and to investigate its clinical significance in CaP. METHODS: Total RNA was extracted from 212 prostatic tissue samples (101 BPH and 111 CaP) and, following cDNA synthesis, quantitative real-time PCR (qPCR) was performed for the expressional quantification of RNase κ. Extensive statistical analysis, including bootstrap resampling, was performed to investigate the differential expression of RNase κ in patients with BPH and CaP and its associations with patients' clinicopathological and survival data. RESULTS: RNase κ was significantly downregulated (P = 0.002) in CaP patients compared to BPH ones. RNase κ overexpression was associated with decreased risk of CaP development and can discriminate between CaP and BPH independently of serum PSA levels (crude odds ratio = 0.93, P = 0.001). RNase κ upregulation was also associated with less advanced (P = 0.018) and less aggressive (P = 0.001) tumors as well as with longer progression-free survival (PFS) (P = 0.003). Finally univariate bootstrap Cox regression confirmed that RNase κ was associated with favorable prognosis (HR = 0.85, P = 0.002). CONCLUSIONS: RNase κ is a biomarker of favorable prognosis in CaP, which is significantly associated with less advanced and aggressive disease, as well as with enhanced PFS.

Effect of cytostatic drugs on the mRNA expression levels of ribonuclease κ in breast and ovarian cancer cells.

Breast and ovarian cancers remain a major public health issue, constituting a major cause of female morbidity and mortality worldwide. Even though systemic chemotherapy remains the main course of action for both types of cancer, current research studies aim at the discovery of novel therapeutic targets. Ribonucleases, due to their apparent implication in gene expression regulation, may serve as potential anticancer drugs. Human RNase κ is an endoribonuclease belonging in a recently identified family of proteins. Recent data from expression microarray studies reveal that the RNase κ gene is found either up- or downregulated in a number of human cancers, indicating a possible diagnostic and/or prognostic utility.The aim of this study was to investigate modulations in the expression levels of RNase κ gene, in breast and ovarian cancer cells, as a response to treatment with different chemotherapeutic agents. BT-20 breast cancer cells and SKOV-3 ovarian cancer cells were treated with the cytotoxic drugs paclitaxel, docetaxel, cisplatin, carboplatin, epirubicin and vinorelbine. Gene expression analysis was performed by the comparative CT method also known as 2(-ΔΔCT) method. The results revealed a distinct increase in the expression levels of RNase κ mRNA (up to 9-fold) after treatment with the antineoplastic agent paclitaxel in both cell lines, while treatment with the remaining anticancer drugs did not alter drastically the mRNA levels of RNase κ. Based on the fact that paclitaxel exerts its cytotoxic action by inducing apoptosis, the results could be indicative of a potential implication of RNase κ in apoptosis-related pathways.

Essential cysteine residues for human RNase κ catalytic activity.

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.

Molecular cloning and characterization of the human RNase kappa, an ortholog of Cc RNase.

A novel protein family, designated hereafter as RNase kappa (kappa) family, has been recently introduced with the characterization of the specific Cc RNase, isolated from the insect Ceratitis capitata. The human ortholog of this family consists of 98 amino acids and shares > 98% identity with its mammalian counterparts. This RNase is encoded by a single-copy gene found to be expressed in a wide spectrum of normal and cancer tissues. The cDNA of the human ribonuclease has been isolated and subcloned into a variety of prokaryotic expression vectors, but most efforts to express it caused a severe toxic effect. On the other hand, the expression of the human RNase by the use of the methylotrophic yeast Pichia pastoris system resulted in the production of a highly active recombinant enzyme. Using a 30-mer 5'-end-labeled RNA probe as substrate, the purified enzyme seems to preferentially cleave ApU and ApG phosphodiester bonds, while it hydrolyzes UpU bonds at a lower rate. Based on amino acid sequence alignment and substrate specificity data, as well as the complete resistance of the recombinant protein to the placental ribonuclease inhibitor, we concluded that the human RNase kappa is a novel endoribonuclease distinct from other known ribonucleases.

Intramolecular disulfide bonding is essential for betanodavirus coat protein conformation.

Here we report on the conformational changes that are responsible for the appearance of the Dicentrarchus labrax encephalitis virus (DlEV) coat protein as a doublet in SDS-PAGE. Wild-type and mutated forms of the coat protein cDNA were expressed in E. coli. The study of the resulting recombinant molecules excluded the possibility of the involvement of a precursor autocatalysis mechanism or a ribosomal frameshifting event in the doublet formation. The appearance of the coat protein doublet was found to be beta-mercaptoethanol sensitive. Based on this observation, we carried out substitution of all cysteine residues. The obtained results demonstrated the importance of intramolecular disulfide bonding between cysteines 187 and 201 on coat protein conformational changes.