27 research outputs found
Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells
AbstractPiwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies
Ring finger protein 126 (RNF126) suppresses ionizing radiation-induced p53-binding protein 1 (53BP1) focus formation
Cells have evolved sophisticated mechanisms to maintain genomic integrity in response to DNA damage. Ionizing radiation (IR)-induced DNA damage results in the formation of IR-induced foci (iRIF) in the nucleus. The iRIF formation is part of the DNA damage response (DDR), which is an essential signaling cascade that must be strictly regulated because either the loss of or an augmented DDR leads to loss of genome integrity. Accordingly, negative regulation of the DDR is as critical as its activation. In this study, we have identified ring finger protein 126 (RNF126) as a negative regulator of the DDR from a screen of iRIF containing 53BP1. RNF126 overexpression abolishes not only the formation of 53BP1 iRIF but also of RNF168, FK2, RAP80, and BRCA1. However, the iRIF formation of H2AX, MDC1, and RNF8 is maintained, indicating that RNF126 acts between RNF8 and RNF168 during the DDR. In addition, RNF126 overexpression consistently results in the loss of RNF168-mediated H2A monoubiquitination at lysine 13/15 and inhibition of the non-homologous end joining capability. Taken together, our findings reveal that RNF126 is a novel factor involved in the negative regulation of DDR, which is important for sustaining genomic integrity
Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems
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the Creative Commons license, and indicate if changes were made.Abstract
Background
Biomarker-driven targeted therapy, the practice of tailoring patients treatment to the expression/activity levels of disease-specific genes/proteins, remains challenging. For example, while the anti-ERBB2 monoclonal antibody, trastuzumab, was first developed using well-characterized, diverse in vitro breast cancer models (and is now a standard adjuvant therapy for ERBB2-positive breast cancer patients), trastuzumab approval for ERBB2-positive gastric cancer was largely based on preclinical studies of a single cell line, NCI-N87. Ensuing clinical trials revealed only modest patient efficacy, and many ERBB2-positive gastric cancer (GC) patients failed to respond at all (i.e., were inherently recalcitrant), or succumbed to acquired resistance.
Method
To assess mechanisms underlying GC insensitivity to ERBB2 therapies, we established a diverse panel of GC cells, differing in ERBB2 expression levels, for comprehensive in vitro and in vivo characterization. For higher throughput assays of ERBB2 DNA and protein levels, we compared the concordance of various laboratory quantification methods, including those of in vitro and in vivo genetic anomalies (FISH and SISH) and xenograft protein expression (Western blot vs. IHC), of both cell and xenograft (tissue-sectioned) microarrays.
Results
The biomarker assessment methods strongly agreed, as did correlation between RNA and protein expression. However, although ERBB2 genomic anomalies showed good in vitro vs. in vivo correlation, we observed striking differences in protein expression between cultured cells and mouse xenografts (even within the same GC cell type). Via our unique pathway analysis, we delineated a signaling network, in addition to specific pathways/biological processes, emanating from the ERBB2 signaling cascade, as a potential useful target of clinical treatment. Integrated analysis of public data from gastric tumors revealed frequent (10 – 20 %) amplification of the genes NFKBIE, PTK2, and PIK3CA, each of which resides in an ERBB2-derived subpathway network.
Conclusion
Our comprehensive bioinformatics analyses of highly heterogeneous cancer cells, combined with tumor omics profiles, can optimally characterize the expression patterns and activity of specific tumor biomarkers. Subsequent in vitro and in vivo validation, of specific disease biomarkers (using multiple methodologies), can improve prediction of patient stratification according to drug response or nonresponse
Effects of Murine Dermal Cells on the Regulation of Hair Growth Is Dependent on the Cell Number and Post-Natal Age of Newborn Mice
Dermal cells from neonatal mice can initiate the formation of hair follicles (HFs) when combined with adult mouse epidermal cells and transplanted subcutaneously into athymic mice. In the present study, the effects of dermal cells on HF formation were tested in terms of total cell number and the time course of cell harvest. Results demonstrated that the number of dermal cells is critical to the formation of HF. Furthermore, hair forming ability is rapidly decreasing as the neonatal mice age. To examine potential differences in gene expression, cDNA array was performed. Results demonstrate that numerous molecules which are directly involved in receptor and signaling correlated with decreased hair inductivity in early time points after delivery. It is reported that bone morphogenic protein (BMP)-6 and Wnt3a treatment increased hair inductivity of dermal papilla cells. But in our study, no changes were observed in the expression levels of BMP-6 and Wnt3a. However, several Wnt related genes demonstrate increased or decreased expression levels. Thus, our results suggest that co-ordinated regulation of these molecules will be important in hair neogenesis within our model system
Hypomorphic Mutations in TONSL Cause SPONASTRIME Dysplasia
SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based as-says and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.Peer reviewe
Targeting Non-Oncogene Addiction for Cancer Therapy
While Next-Generation Sequencing (NGS) and technological advances have been useful in identifying genetic profiles of tumorigenesis, novel target proteins and various clinical biomarkers, cancer continues to be a major global health threat. DNA replication, DNA damage response (DDR) and repair, and cell cycle regulation continue to be essential systems in targeted cancer therapies. Although many genes involved in DDR are known to be tumor suppressor genes, cancer cells are often dependent and addicted to these genes, making them excellent therapeutic targets. In this review, genes implicated in DNA replication, DDR, DNA repair, cell cycle regulation are discussed with reference to peptide or small molecule inhibitors which may prove therapeutic in cancer patients. Additionally, the potential of utilizing novel synthetic lethal genes in these pathways is examined, providing possible new targets for future therapeutics. Specifically, we evaluate the potential of TONSL as a novel gene for targeted therapy. Although it is a scaffold protein with no known enzymatic activity, the strategy used for developing PCNA inhibitors can also be utilized to target TONSL. This review summarizes current knowledge on non-oncogene addiction, and the utilization of synthetic lethality for developing novel inhibitors targeting non-oncogenic addiction for cancer therapy
RNF126, 168 and CUL1: The Potential Utilization of Multi-Functional E3 Ubiquitin Ligases in Genome Maintenance for Cancer Therapy
Ubiquitination is a post-translational modification (PTM) that is involved in proteolysis, protein–protein interaction, and signal transduction. Accumulation of mutations and genomic instability are characteristic of cancer cells, and dysfunction of the ubiquitin pathway can contribute to abnormal cell physiology. Because mutations can be critical for cells, DNA damage repair, cell cycle regulation, and apoptosis are pathways that are in close communication to maintain genomic integrity. Uncontrolled cell proliferation due to abnormal processes is a hallmark of cancer, and mutations, changes in expression levels, and other alterations of ubiquitination factors are often involved. Here, three E3 ubiquitin ligases will be reviewed in detail. RNF126, RNF168 and CUL1 are involved in DNA damage response (DDR), DNA double-strand break (DSB) repair, cell cycle regulation, and ultimately, cancer cell proliferation control. Their involvement in multiple cellular pathways makes them an attractive candidate for cancer-targeting therapy. Functional studies of these E3 ligases have increased over the years, and their significance in cancer is well reported. There are continuous efforts to develop drugs targeting the ubiquitin pathway for anticancer therapy, which opens up the possibility for these E3 ligases to be evaluated for their potential as a target protein for anticancer therapy
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Defining the role of Mtf1 and N-terminal domain of Rpo41 in transcription initiation and replication
textMitochondrion is an organelle found in the eukaryotic cell. It is responsible for essential metabolic processes as well as ATP production via oxidative phosphorylation (OXPHOS). The mitochondrion contains DNA that encodes for several subunits in the OXPHOS system as well as rRNA and tRNA for translation. It also has its own replication, transcription and translation machinery. Proper maintenance of the mitochondrial DNA is critical for the cell’s health.
Saccharomyces cerevisiae mitochondrial transcription system has been a great model system for its ease of genetic manipulation as well as having conserved RNA polymerases across species. The polymerases are homologues to T7 RNA polymerase, but have longer N-terminal domain and require transcription factor(s). The reason for the extra domain as well as the need for an accessory factor is still unclear. This study reveals the role of Rpo41 N-terminal domain (NTD) as well as clarifies the role of Mtf1, the transcription factor, in transcription initiation.
Rpo41 is the 153 kDa catalytic subunit, and Mtf1 is 40 kDa, the transcription factor of the yeast mitochondria. We have shown that Mtf1 is required for correct promoter sequence recognition as well as inhibition of incorrect initiation. Although it was thought that Rpo41 has intrinsic promoter recognition capability, we have shown that Rpo41 can initiate transcription on a pre-melted DNA, even if it is not the consensus promoter sequence. N-terminal truncation mutant studies showed that the NTD of Rpo41 is also required for correct transcription initiation. On linear duplex DNA, N-terminal truncation of 321 amino acids has little effect when Mtf1 is present. On pre-melted DNA, it shows opposite trend from the wild-type. 160 N-terminal amino acid residue truncation shows little activity, whereas Mtf1 increases activity, even on non-promoter initiation sites.
We further investigated properties of Rpo41 in replication. A link between mitochondrial transcription and replication has been suggested before, where Rpo41 functions as the leading strand primase. Our studies show that Rpo41 can indeed function as the leading and lagging strand primase, and explains why Rpo41 is able to initiate transcription on non-promoter sites. N-terminal truncation resulted in loss of primase activity, which shows that NTD is required for replication.Cellular and Molecular Biolog
Yeast Mitochondrial Transcription Factor Mtf1 Determines the Precision of Promoter-Directed Initiation of RNA Polymerase Rpo41
<div><p>Despite their clear T7-bacteriophage origin, mitochondrial RNA polymerases have evolved to require transcription factors. All mitochondrial polymerases contain an extra N-terminal domain that has no counterpart in the self-proficient phage enzyme, which is therefore hypothesized to interact with transcription factors. We studied a series of N-terminal deletion mutants of yeast mitochondrial RNA polymerase, Rpo41, and have found that the N-terminal region does not abolish the effects of Mtf1; rather it contributes directly to enzyme catalysis. Mtf1 can rescue the defective Rpo41 enzymes resulted from N-terminal domain deletions. Although Rpo41 appears to have retained all promoter recognition elements found in T7 RNAP, the elements are not independently functional, and Mtf1 is necessary and sufficient for holoenzyme promoter-directed transcription activity.</p></div
Interactions of Rpo41 with Mtf1 and promoter DNA.
<p>(A). Analytical gel filtration chromatograph of wild-type Rpo41 (2.8 μM) with Mtf1 (5.6 μM) (red) superimposed with that of Rpo41 (1 μM), Mtf1 (2 μM) and a 20bp promoter-containing DNA duplex (3 μM) (blue). The elution volume of Rpo41 alone is indicated (black arrow). The protein contents of the peak fractions of Rpo41 + Mtf1 and Rpo41 + Mtf1 + DNA were visualized after SDS-PAGE. (B). Analyses described in (A) performed with Rpo41 ΔN393.</p