Effects of folate on telomere length and chromosome stability of human fibroblasts and melanoma cells in vitro: a comparison of folic acid and 5-methyltetrahydrofolate.

Telomere length (TL), which is maintained by human telomerase reverse transcriptase (hTERT; component of telomerase) and/or TRF1/TRF2 (core components of shelterin) via different mechanisms, is essential for chromosomal stability and cell survival. Folates comprise a group of essential B9 vitamin that involve in DNA synthesis and methylation. This study aimed to evaluate the effects of folic acid (FA) and 5-methyltetrahydrofolate (5-MeTHF) on TL, chromosome stability, and cell survival of telomerase-negative BJ and telomerase-positive A375 cells in vitro. BJ and A375 cells were cultured in modified medium with FA or 5-MeTHF (22.6 or 2260 nM) for 28 days. TL and mRNA expression were determined by RT-qPCR. Chromosome instability (CIN) and cell death were measured by CBMN-Cyt assay. Results showed that abnormal TL elongation was observed in FA- and 5-MeTHF-deficient BJ cells. The TL of A375 cells showed no obvious alterations under the FA-deficient condition but was significantly elongated under the 5-MeTHF-deficient condition. In both BJ and A375 cells, FA and 5-MeTHF deficiency caused decreased TRF1, TRF2, and hTERT expression, increased CIN and cell death; while a high concentration of 5-MeTHF induced elongated TL, elevated CIN, increased TRF1 and TRF2 expression, and decreased hTERT expression, when compared with the FA counterpart. These findings concluded that folate deficiency induced TL instability in both telomerase-negative and -positive cells, and FA was more efficient in maintaining TL and chromosome stability compared with 5-MeTHF.

Effects of folate on telomere length and chromosome stability of human fibroblasts and melanoma cells in vitro: a comparison of folic acid and 5-methyltetrahydrofolate.

Telomere length (TL), which is maintained by human telomerase reverse transcriptase (hTERT; component of telomerase) and/or TRF1/TRF2 (core components of shelterin) via different mechanisms, is essential for chromosomal stability and cell survival. Folates comprise a group of essential B9 vitamin that involve in DNA synthesis and methylation. This study aimed to evaluate the effects of folic acid (FA) and 5-methyltetrahydrofolate (5-MeTHF) on TL, chromosome stability, and cell survival of telomerase-negative BJ and telomerase-positive A375 cells in vitro. BJ and A375 cells were cultured in modified medium with FA or 5-MeTHF (22.6 or 2260 nM) for 28 days. TL and mRNA expression were determined by RT-qPCR. Chromosome instability (CIN) and cell death were measured by CBMN-Cyt assay. Results showed that abnormal TL elongation was observed in FA and 5-MeTHF deficient BJ cells. The TL of A375 cells showed no obvious alterations under the FA-deficient condition but was significantly elongated under the 5-MeTHF-deficient condition. In both BJ and A375 cells, FA and 5-MeTHF deficiency caused decreased TRF1, TRF2, and hTERT expression, increased CIN and cell death; while a high concentration of 5-MeTHF induced elongated TL, elevated CIN, increased TRF1 and TRF2 expression and decreased hTERT expression, when compared with the FA counterpart. These findings concluded that folate deficiency induced TL instability in both telomerase-negative and -positive cells, and FA was more efficient in maintaining TL and chromosome stability compared with 5-MeTHF.

Effects of dietary restriction on genome stability are sex and feeding regimen dependent.

Preserving genome stability is essential to prevent aging and cancer. Dietary restriction (DR) is the most reproducible non-pharmacological way to improve health and extend lifespan in various species. Whether DR helps to preserve genome stability and whether this effect is altered by experimental variables remain unclear. Moreover, DR research relies heavily on experimental animals, making the development of reliable in vitro mimetics of great interest. Therefore, we tested the effects of sex and feeding regimen (time-restricted eating, alternate day fasting and calorie restriction) on genome stability in CF-1 mice and whether these effects can be recapitulated by cell culture paradigms. Here, we show that calorie restriction significantly decreases the spontaneous micronuclei (MN), a biomarker of genome instability, in bone marrow cells of females instead of males. Alternate day fasting significantly decreases cisplatin-induced MN in females instead of males. Unexpectedly, daily time-restricted eating significantly exacerbates cisplatin-induced MN in males but not in females. Additionally, we design several culture paradigms that are able to faithfully recapitulate the key effects of these DR regimens on genome stability. In particular, 30% reduction of serum, a mimetic of calorie restriction, exhibits a strong ability to decrease spontaneous and cisplatin-induced MN in immortalized human umbilical vein endothelial cells. We conclude that the effects of different DR regimens on genome stability are not universal and females from each diet regimen sustain a more stable genome than males. Our results provide novel insight into the understanding of how DR influences genome stability in a sex and regimen dependent way, and suggest that our in vitro DR mimetics could be adopted to study the underlying molecular mechanisms.

Interplay of cGAS with micronuclei: Regulation and diseases.

In higher eukaryotes, sophisticate regulation of genome function requires all chromosomes to be packed into a single nucleus. Micronucleus (MN), the dissociative nucleus-like structure frequently observed in aging and multiple disease settings, has critical, yet under-recognized, pathophysiological functions. Micronuclei (MNi) have recently emerged as major sources of cytosolic DNA that can activate the cGAS-STING axis in a cell-intrinsic manner. However, MNi induced from different genotoxic stressors display great heterogeneity in binding or activating cGAS and the signaling responses downstream of the MN-induced cGAS-STING axis have divergent outcomes including autoimmunity, autoinflammation, metastasis, or cell death. Thus, full characterization of molecular network underpinning the interplay of cGAS and MN is important to elucidate the pathophysiological roles of immunogenic MN and design improved drugs that selectively target cancer via boosting the MN-derived cGAS-STING axis. Here, we summarize our current understanding of the mechanisms for self-DNA discrimination by cGAS. We focus on discussing how MN immunogencity is dictated by multiple mechanisms including integrity of micronuclear envelope, state of nucleosome and DNA, competitive factors, damaged mitochondrial DNA and micronucleophagy. We also describe emerging links between immunogenic MN and human diseases including cancer, neurodegenerative diseases and COVID-19. Particularly, we explore the exciting concept of inducing immunogenic MN as a therapeutic approach in treating cancer. We propose a new theoretical framework to describe immunogenic MN as a biological sensor to modulate cellular processes in response to genotoxic stress and provide perspectives on developing novel experimental approaches to unravel the complexity of MN immunogenicity regulation and immunogenic MN pathophysiology.

MicroRNA-22-3p and MicroRNA-149-5p Inhibit Human Hepatocellular Carcinoma Cell Growth and Metastasis Properties by Regulating Methylenetetrahydrofolate Reductase.

microRNAs are small endogenous noncoding RNAs that have emerged as key negative regulators that target gene expression through RISC. Our previous study showed that the methylenetetrahydrofolate reductase gene (MTHFR) plays a key role in one carbon metabolism, which is downregulated by miR-22-3p and miR-149-5p, and that it could exert a potential anti-cancer effect. Whether miR-22-3p/miR-149-5p can regulate MTHFR to exert anti-cancer effects has become the focus of our research. Normal (HL-7702 cells) and cancerous (QGY-7703/HepG2 cells) human hepatocellular cells were transfected with 100 nM hsa-miR-22-3p/hsa-miR-149-5p mimic or controls. After 24, 48, and 72 h, cell proliferation ability was tested using CCK-8. The changes in MTHFR expression at both the transcriptional and translational levels were determined by RT-qPCR and Western blotting, respectively. Cancerous cell invasion and migration ability were confirmed by means of a transwell assay. We found that ectopic miR-22-3p/miR-149-5p inhibits hepatocellular carcinoma cell proliferation but does not inhibit normal human hepatocyte proliferation. The transfection of ectopic miR-22-3p/miR-149-5p downregulated the MTHFR expression in QGY-7703 and HepG2 but not in HL-7702. QGY-7703 and HepG2 migration and invasion were inhibited by ectopic miR-22-3p/miR-149-5p. Additionally, we found that ectopic miR-22-3p/miR-149-5p significantly increased the expression of TP53INP1 and PDCD4 in QGY-7703. The results of the study suggest that miRNA-22-3p and miRNA-149-5p inhibit tumor growth and metastasis properties may be by regulating MTHFR and that they exert anticancer effects in hepatocellular carcinoma cells.

Loss of Y chromosome: An emerging next-generation biomarker for disease prediction and early detection?

As human life expectancy increases substantially and aging is the primary risk factor for most chronic diseases, there is an urgent need for advancing the development of post-genomic era biomarkers that can be used for disease prediction and early detection (DPED). Mosaic loss of Y chromosome (LOY) is the state of nullisomy Y in sub-groups of somatic cells acquired from different post-zygotic development stages and onwards throughout the lifespan. Multiple large-cohort based epidemiology studies have found that LOY in blood cells is a significant risk factor for future mortality and various diseases in males. Many features intrinsic to LOY analysis may be leveraged to enhance its use as a non-invasive, sensitive, reliable, high throughput-biomarker for DPED. Here, we review the emerging literatures in LOY studies and highlight ten strengths for using LOY as a novel biomarker for genomics-driven DPED diagnostics. Meanwhile, the current limitations in this area are also discussed. We conclude by identifying some important knowledge gaps regarding the consequences of malsegregation of the Y chromosome and propose further steps that are required before clinical implementation of LOY. Taken together, we think that LOY has substantial potential as a biomarker for DPED, despite some hurdles that still need to be addressed before its integration into healthcare becomes acceptable.

Understanding the birth of rupture-prone and irreparable micronuclei.

Micronuclei are extra-nuclear bodies mainly derived from ana-telophase lagging chromosomes/chromatins (LCs) that are not incorporated into primary nuclei at mitotic exit. Unlike primary nuclei, most micronuclei are enclosed by nuclear envelope (NE) that is highly susceptible to spontaneous and irreparable rupture. Ruptured micronuclei act as triggers of chromothripsis-like chaotic chromosomal rearrangements and cGAS-mediated innate immunity and inflammation, raising the view that micronuclei play active roles in human aging and tumorigenesis. Thus, understanding the ways in which micronuclear envelope (mNE) goes awry acquires increased importance. Here, we review the data to present a general framework for this question. We firstly describe NE reassembly after mitosis and NE repair during interphase. Simultaneously, we briefly discuss how mNE is organized and how mNE rupture controls the fate of micronuclei and micronucleated cells. As a focus of this review, we highlight current knowledge about why mNE is rupture-prone and irreparable. For this, we survey observations from a series of elegant studies to provide a systematic overview. We conclude that the birth of rupture-prone and irreparable micronuclei may be the cumulative effects of their intracellular geographic origins, biophysical properties, and specific mNE features. We propose that DNA damage and immunogenicity in micronuclei increase stepwise from altered mNE components, mNE rupture, and refractory to repair. Throughout our discussion, we note interesting issues in mNE fragility that have yet to be resolved.

Aqueous extract of bulbus Fritillaria cirrhosa induces cytokinesis failure by blocking furrow ingression in human colon epithelial NCM460 cells.

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.

Mosaic loss of human Y chromosome: what, how and why.

Y chromosome (ChrY), the male-specific sex chromosome, has been considered as a genetic wasteland. Aging-related mosaic loss of ChrY (LOY) has been known for more than half a century, but it was constantly considered as a neutral karyotype related to normal aging. These views have been challenged with genome-wide association studies identifying mosaic LOY in human somatic cells is the most commonly acquired mutation in male's genome and is associated with a wide spectrum of human diseases including cancer, Alzheimer's disease, and cardiovascular disease. These previously undescribed clinical significances deeply modify our perception on ChrY and open up a range of new questions. Here, we review the latest advances in our knowledge of the biological origins and clinical consequences of mosaic LOY. We highlight the association of mosaic LOY to pathogenic conditions and evaluate the cause-and-consequence relationships between mosaic LOY and pathogenesis. The known risk factors of mosaic LOY including age, genetic variants, ChrY structural aberrations and environmental stressors are discussed. In light of evidence from pioneering and more recent studies, we propose the micronucleation hypothesis and centromere-dysfunction and telomere-attrition models to explain how mosaic LOY occurs and why ChrY is prone to lose. We believe it is importantly and timely to extend mosaic LOY research from epidemiological associations to mechanistic studies. In this regard, we outline important gaps and assess several future directions from a biological and clinical perspective. An improved understanding of mosaic LOY will open new pathways to modify and increase healthy aging in males.

The molecular origins and pathophysiological consequences of micronuclei: New insights into an age-old problem.

Micronuclei (MN), the small nucleus-like bodies separated from the primary nucleus, can exist in cells with numerical and/or structural chromosomal aberrations in apparently normal tissues and more so in tumors in humans. While MN have been observed for over 100 years, they were merely and constantly considered as passive indicators of chromosome instability (CIN) for a long time. Relatively little is known about the molecular origins and biological consequences of MN. Rapid technological advances are helping to close these gaps. Very recent studies provide exciting evidence that MN act as key platform for chromothripsis and a trigger of innate immune response, suggesting that MN could affect cellular functions by both genetic and nongenetic means. These previously unappreciated findings have reawakened widespread interests in MN. In this review, the diverse mechanisms leading to MN generation and the complex fate profiles of MN are discussed, together with the evidence for their contribution to CIN, inflammation, senescence and cell death. Moreover, we put this knowledge together into a speculative perspective on how MN may be responsible for cancer development and how their presence may influence the choice of treatment. We suggest that the heterogeneous responses to MN may function physiological to ensure the arrestment, elimination and immune clearance of damaged cells, but pathologically, may enable the survival and oncogenic transformation of cells bearing CIN. These insights not only underscore the complexity of MN biology, but also raise a host of new questions and provide fertile ground for future research.

High concentration of sugars is genotoxic to folate-deficient cells.

Patients with type 2 diabetes mellitus (T2DM) are associated with an elevated, but poorly understood baseline of genomic instability (GIN). Expert panels are still debating on whether hyperglycemia is the key element in conferring this high GIN. Since high blood glucose and low blood folate are prevalent in T2DM, we hypothesized that high glucose may work with low folate to induce GIN. Using NCM460, CCD841 and L02 cell lines as in vitro cell models, we investigated the genotoxic effects of high sugars (HS; 1-2% glucose, fructose, galactose or sucrose) alone or in combination with folate deficiency (23 nM, FD) over a course of 7 days by the cytokinesis block micronucleus assay. We found that HS is nongenotoxic to NCM460, CCD841 and L02 cells. However, the combination of HS and FD induced significantly higher levels of micronuclei, nucleoplasmic bridges and nuclear buds. Our in vitro work demonstrates that HS is non-genotoxic under folate repletive condition, but is genotoxic under FD condition. These results provide preclinal proof of concept that concomitant hyperglycemia and low folate may explain, at least in part, the high baseline of GIN in T2DM patients, suggesting that folate levels should be kept under control in order to limit the risk of GIN and carcinogenesis in T2DM.

Geraniin Differentially Modulates Chromosome Stability of Colon Cancer and Noncancerous Cells by Oppositely Regulating their Spindle Assembly Checkpoint.

Geraniin has been reported to specifically induce apoptosis in multiple human cancers, but the underlying mechanism is poorly defined. The spindle assembly checkpoint (SAC) is a surveillance system to ensure high-fidelity chromosome segregation during mitosis. Weakening of SAC to enhance chromosome instability (CIN) can be therapeutic because very high levels of CIN are lethal. In this study, we have investigated the effects of geraniin on the SAC of colorectal cancer HCT116 cells and noncancerous colon epithelial CCD841 cells. We find that treatment of HCT116 cells with geraniin leads to dose-dependent decrease of cell proliferation, colony formation, and anchorage-independent growth. Geraniin is found to induce apoptosis in mitotic and postmitotic HCT116 cells. Furthermore, geraniin weakens the SAC function of HCT116 cells by decreasing the transcriptional expression of several SAC kinases (particularly Mad2 and Bub1), which in turn leads to premature anaphase entry, mitotic aberrations, and CIN in HCT116 cells. In contrast, the proliferation of CCD841 cells is slightly inhibited by geraniin. Even more interestingly, geraniin increases the transcriptional expression of several SAC kinases (e.g., Mad1 and BubR1) to strengthen SAC efficiency, which contributes to the reduction of mitotic aberrations and CIN in CCD841 cells. Altogether, our findings reveal that the SAC pathway in human colon cancer and noncancerous cell lineages responses oppositely to geraniin treatment, resulting CIN promotion and suppression, respectively. Specific abrogation of SAC to induce catastrophic CIN in HCT116 cells may account for the selective anticancer action of geraniin.. Environ. Mol. Mutagen. 60:254-268, 2019. © 2018 Wiley Periodicals, Inc.

Geraniin selectively promotes cytostasis and apoptosis in human colorectal cancer cells by inducing catastrophic chromosomal instability.

Homeostasis of chromosomal instability (CIN) facilitates the origin and evolution of abnormal karyotypes that are critical for the survival and proliferation of cancer cells, but excessive CIN can result in cellular toxicity. Geraniin is a multifunctional ellagitannin found in some species of Geranium and Phyllanthus. We employed the cytokinesis-block micronucleus cytome assay to evaluate the CIN, nuclear division index (NDI) and apoptosis induced by geraniin in human colorectal adenocarcinoma cells (Colo205 and Colo320) and human colon mucosal epithelial cells (NCM460). Cells were exposed to 25, 50 or 100 µg/ml geraniin for 24, 48 or 72 h. 0.05 µg/ml mitomycin C was used as a positive control and media as a negative control. The results showed that, compared to negative controls, geraniin significantly reduced NDI (P < 0.01) and increased CIN (P < 0.01) and apoptosis (P < 0.05) in Colo205 and Colo320 cells in a dose- and time-dependent manner. Conversely, geraniin significantly increased NDI (P < 0.05) and decreased CIN (P < 0.001) and apoptosis (P < 0.01) in NCM460 cells. Moreover, CIN was positively associated with apoptosis (r = 0.437, P < 0.001) and negatively associated with NDI (r = -0.744, P < 0.001) in these cells. Together, our results highlight that the induction of catastrophic CIN may underlie the antitumor potential of geraniin. Our data also suggest that geraniin can decrease the risk of oncogenic transformation via decreasing CIN in normal cells.

Shelterin differentially respond to oxidative stress induced by TiO2-NPs and regulate telomere length in human hepatocytes and hepatocarcinoma cells in vitro.

Titanium dioxide nanoparticles (TiO2-NPs) have raised serious attention for their widely use and potential adverse effects on human mainly due to producing ROS. However, the influence of TiO2-NPs on telomere maintaining has not been studied clearly. Shelterin plays core roles in telomere length (TL) regulation. Abnormal TL are associated with chromosome instability (CIN) and high risk of diseases. This study investigated whether TiO2-NPs affect TL to induce CIN through ROS generation and the possible mechanisms. Human hepatocyte L-02 and hepatocarcinoma cells QGY were exposed to TiO2-NPs (0, 40, 80 µg/mL) for 72 h. The intracellular hydrogen dioxide (H2O2) concentration were measured. The TL, Nrf-2, and three core shelterin components (TRF1, TRF2, and POT1) transcription level were determined by quantitative real-time PCR. CIN was measured by cytokinesis-block micronucleus assay. TiO2-NPs exposure increased intracellular H2O2 in both L-02 and QGY cells, and induced Nrf-2, TRF1, TRF2, POT1 downregulated transcription compared with control (P < 0.001) in L-02 but all upregulated (P < 0.05) in QGY. Significant TL shortening (P < 0.001) and CIN increase (P < 0.01) in L-02 cells were observed but not in QGY cells. The differentially responses of the tested components of shelterin and Nrf-2 to oxidative stress induced by TiO2-NPs led to the weakened telomere protection in normal cells and effective telomere maintenance in cancer cells, respectively. The upregulation of Nrf-2 and shelterin could protect TL and chromosome stability against TiO2-NPs exposure.

Biphasic regulation of spindle assembly checkpoint by low and high concentrations of resveratrol leads to the opposite effect on chromosomal instability.

Resveratrol (RSV) is a naturally occurring polyphenolic phytoalexin possessing numerous health-promoting effects. Chromosomal instability (CIN), usually results from defective spindle assembly checkpoint (SAC), is a major contributor to many diseases. While it's recently recognized that RSV exhibits a nonlinear dose response for disease prevention, whether it's the case for its role in CIN remains unknown. Here, we investigated the potential of a broad range of RSV concentrations (0.01-100µM) on CIN and the underlying mechanisms in human normal colon epithelial NCM460 cells. CIN was measured by cytokinesis-block micronucleus assay; mitotic fidelity was determined by aberrant mitosis analysis; SAC activity was assessed by nocodazole-challenge assay, and the expression of SAC genes was examined by RT-qPCR. We found that 0.1µM RSV significantly reduced CIN (P<0.01), while 100µM RSV significantly induced it (P<0.05). Mitotic infidelity was significantly prevented by 0.1µM RSV but promoted by 100µM RSV (P<0.05 for both). Moreover, the function of SAC was sustained and impaired by 0.1µM and 100µM RSV, respectively. Several SAC genes, including Aurora-B, Aurora-C, Plk-1 and CENP-E, were significantly up-regulated and down-regulated by 0.1µM and 100µM RSV, respectively (P<0.05). In conclusion, RSV exhibited a biphasic dose-dependent effect on CIN that was exerted via the regulation of mitotic fidelity through the SAC network. The health implications of these findings were summarized.

Phyllanthus emblica Linn. fruit extract potentiates the anticancer efficacy of mitomycin C and cisplatin and reduces their genotoxicity to normal cells in vitro.

OBJECTIVE: Fruit of Phyllanthus emblica Linn. (PE) is widely consumed as a functional food and used as a folk medicine due to its remarkable nutritional and pharmacological effects. Mitomycin C (MMC) and cisplatin (cDDP) are the most widely used forms of chemotherapeutic drug, but their clinical use is limited by their genotoxicity to normal cells. We aimed to determine whether PE has potential to reduce the genotoxicity, while improving the anticancer effect, of MMC and cDDP. METHODS: Cell proliferation was evaluated using the trypan blue exclusion assay and colony-forming assay. Genomic instability (GIN) was measured using the cytokinesis-block micronucleus assay. RESULTS: Co-treatment (72 h) with PE at 20-320 µg/ml significantly enhanced the efficacy of MMC (0.05 µg/ml) and cDDP (1 µg/ml) against Colo205 colorectal cancer cells (P<0.05), and at 80-320 µg/ml significantly decreased MMC- and cDDP-induced GIN and multinucleation in normal colonic NCM460 cells (P<0.05). PE significantly decreased the mitotic index (P<0.01), blocked mitotic progression (P<0.05), and promoted apoptosis (P<0.01) in MMC- and cDDP-treated NCM460 cells, suggesting that PE-mediated inhibition of mitosis and induction of apoptosis may limit the division and survival of highly damaged cells. Also, PE was found to inhibit the clonal expansion of MMC- and cDDP-treated NCM460 cells (P<0.05) and decrease the heterogeneity of the surviving clones. CONCLUSIONS: PE potentiates the anticancer efficacy of MMC and cDDP, while preventing their genotoxicity and inhibiting clonal expansions of unstable genomes in normal cells. These data suggest that PE has the potential to reduce the risk of secondary cancers induced by chemotherapeutics.

Folate deficiency induces mitotic aberrations and chromosomal instability by compromising the spindle assembly checkpoint in cultured human colon cells.

Folates comprise the essential B9 vitamin that act as cofactors and cosubstrates in one-carbon metabolism for both biosynthesis and methylation of DNA and RNA. Folate deficiency (FD) has been shown to induce chromosomal instability (CIN), yet the underlying mechanisms are poorly understood. Here, we used human NCM460 colon mucosal cells as a model to investigate the effect of FD on spindle assembly checkpoint (SAC), a cell-cycle regulatory pathway preventing CIN during mitosis. Cells were maintained in medium containing 1.36 (FD) and 2260 nM (control, FC) folate for 21 days. CIN was measured by cytokinesis-block micronucleus assay; mitotic infidelity was determined by aberrant mitosis analysis; SAC activity was assessed by nocodazole-challenge assay, and the expression of core SAC genes was examined by real-time quantitative PCR (RT-qPCR). We found that, relative to FC, FD significantly induced CIN in a time-dependent way (P < 0.01). Mitotic cells cultured in FD medium had significant higher frequencies of misalignment, misegregation and spindle multipolarity than those cultured in FC medium (P < 0.01). FD-induced SAC impairment and overriding, resulting premature mitotic exit and cell multinucleation (P < 0.05). Moreover, FD deregulated the expression of several key SAC genes (P < 0.01). Overall, these data are the first to demonstrate that FD substantially compromises SAC network which predisposes cells to mitotic aberrations and CIN. These results establish a new link between folate metabolism and SAC signalling, two pathways that are highly relevant for tumorigenesis.

Association between the MTHFR C677T polymorphism, blood folate and vitamin B12 deficiency, and elevated serum total homocysteine in healthy individuals in Yunnan Province, China.

BACKGROUND: An increased serum total homocysteine (tHcy) concentration is typically associated with genetic defects involved in Hcy metabolism or related nutritional deficiencies. In this study, the combined effects of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and folate and vitamin B12 deficiency on serum total Hcy (tHcy) levels were evaluated in a healthy Chinese population in Yunnan Province, China. METHODS: The MTHFR C677T polymorphism was genotyped in 330 volunteers (164 men and 166 women) using polymerase chain reaction-restriction fragment length polymorphism analysis. Folate, vitamin B12, and tHcy concentrations were determined by corpuscle immune chemiluminescence assays. The tHcy concentration was determined using an enzymatic assay. RESULTS: Significant negative correlations (p<0.001) were observed between the serum levels of tHcy and folate (r=-0.252) and vitamin B12 (r=-0.243). Men had significantly higher serum tHcy concentrations than women (p<0.001). Individuals with the MTHFR TT genotype had significantly higher serum tHcy concentrations than individuals with the CC and CT genotypes (p<0.001). The folate level of red blood cells was significantly increased in individuals with the TT genotype than in individuals with the CC genotype (p<0.05). Moreover, in the low vitamin group, the serum tHcy level was significantly correlated with the levels of folate (r=-0.334, p=0.001) and vitamin B12 (r=-0.212, p=0.046). CONCLUSION: The MTHFR C677T polymorphism, folate deficiency, and B12 deficiency were significantly associated with elevated serum tHcy levels. Among these three factors, folate deficiency had the greatest contribution to the serum tHcy concentration, followed by (in order of decreasing effect) MTHFR C677T and vitamin B12 deficiency. Thus, folic acid and vitamin B12 supplementation could help prevent diseases associated with tHcy accumulation, especially in individuals with the MTHFR 677TT genotype.

Extract of bulbus Fritillaria cirrhosa perturbs spindle assembly checkpoint, induces mitotic aberrations and genomic instability in human colon epithelial cell line.

BACKGROUND: Bulbus Fritillaria cirrhosa D. Don (BFC) has been used in China as a folk medicine for the treatment of cough and asthma for more than 2000 years. The antitussive and antiasthmatic effects of BFC have been reported before, nevertheless its toxicity and safety have not been documented. This study investigated the possible effects of BFC on spindle assembly checkpoint (SAC), mitotic fidelity and genomic stability in human NCM460 colon epithelial cells. METHODS: Cells were treated with BFC (0, 20, 40, 80 and 160µg/ml) for 24, 48 and 72h and harvested differently according to the biomarkers observed. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment (CMA), chromosome lagging (CL) and chromatin bridge (CB). Frequencies of micronuclei (MN), nucleoplasmic bridge and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of genomic instability (GIN). SAC activity was determined by anaphase to metaphase ratio (AMR) and the expression of several SAC genes, including CENP-E, Mps1, Bub1, Mad-1, BubR1 and Mad-2. RESULTS: Compared with the control, cells in BFC treated groups (80 and 160µg/ml) showed: 1) increased AMR (p<0.05), up-regulated expression of Mps1, Bub1 and Mad-1 (p<0.05) and down-regulated expression of CENP-E, BubR1 and Mad-2 (p<0.05); 2) increased frequencies of CMA, CL and CB (p<0.01); 3) increased incidences of MN and NB (p<0.01). CONCLUSIONS: This study revealed for the first time that BFC causes mitotic aberrations and GIN in human colon epithelial cells and these effects maybe the result of SAC dysfunction.

Gene therapy for hemophilia B mice with scAAV8-LP1-hFIX.

Hemophilia B is a hemorrhagic disease caused by the deficiency of clotting factor IX (FIX). Gene therapy might be the ultimate strategy for the disease. However, two main problems that should be solved in gene therapy for hemophilia B are immunity and safety. Self-complementary adeno-associated virus serotype 8 (scAAV8), a non-human primate AAV featuring low immunogenicity and high transfection efficiency in liver cells, might be a potential vector for hemophilia B gene therapy. A strong liver-specific promoter-1 (LP1) was inserted and mutant human FIX Arg338Ala was introduced into plasmid scAAV8-LP1 to develop an optimized AAV8 vector that expresses human clotting factor FIX (hFIX). The efficiency of scAAV8-LP1-hFIX administered through normal systemic injection or hydrodynamic injection was compared. A high expression was achieved using hydrodynamic injection, and the peak hFIX expression levels in the 5 × 10(11) and 1 × 10(11) virus genome (vg) cohorts were 31.94% and 25.02% of normal level, respectively, at 60 days post-injection. From the perspective of long-term (200 days) expression, both injection methods presented promising results with the concentration value maintained above 4% of normal plasma. The results were further verified by enzyme-linked immunosorbent assay and activated partial thromboplastin time. Our study provides a potential gene therapy method for hemophilia B.