Critical roles of tubular mitochondrial ATP synthase dysfunction in maleic acid-induced acute kidney injury.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400 mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2 mM or 5 mM MA for 24 h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p < 0.005). From the pathological analysis, MA-induced AKI aggravated renal tubular injuries, increased kidney injury molecule-1 (KIM-1) expression and caused renal tubular cell apoptosis. At the cellular level, mitochondrial dysfunction was found with increasing mitochondrial reactive oxygen species (ROS) (p < 0.001), uncoupled mitochondrial respiration with decreasing electron transfer system activity (p < 0.001), and decreasing ATP production (p < 0.05). Under transmission electron microscope (TEM) examination, the cristae formation of mitochondria was defective in MA-induced AKI. To unveil the potential target in mitochondria, gene expression analysis revealed a significantly lower level of ATPase6 (p < 0.001). Renal mitochondrial protein levels of ATP subunits 5A1 and 5C1 (p < 0.05) were significantly decreased, as confirmed by protein analysis. Our study demonstrated that dysfunction of mitochondria resulting from altered expression of ATP synthase in renal tubular cells is associated with MA-induced AKI. This finding provides a potential novel target to develop new strategies for better prevention and treatment of MA-induced AKI.

The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Mitochondrial Signaling, the Mechanisms of AKI-to-CKD Transition and Potential Treatment Targets.

Acute kidney injury (AKI) is increasing in prevalence and causes a global health burden. AKI is associated with significant mortality and can subsequently develop into chronic kidney disease (CKD). The kidney is one of the most energy-demanding organs in the human body and has a role in active solute transport, maintenance of electrochemical gradients, and regulation of fluid balance. Renal proximal tubular cells (PTCs) are the primary segment to reabsorb and secrete various solutes and take part in AKI initiation. Mitochondria, which are enriched in PTCs, are the main source of adenosine triphosphate (ATP) in cells as generated through oxidative phosphorylation. Mitochondrial dysfunction may result in reactive oxygen species (ROS) production, impaired biogenesis, oxidative stress multiplication, and ultimately leading to cell death. Even though mitochondrial damage and malfunction have been observed in both human kidney disease and animal models of AKI and CKD, the mechanism of mitochondrial signaling in PTC for AKI-to-CKD transition remains unknown. We review the recent findings of the development of AKI-to-CKD transition with a focus on mitochondrial disorders in PTCs. We propose that mitochondrial signaling is a key mechanism of the progression of AKI to CKD and potential targeting for treatment.

Dual roles of myocardial mitochondrial AKT on diabetic cardiomyopathy and whole body metabolism.

BACKGROUND: The PI3K/AKT pathway transduces the majority of the metabolic actions of insulin. In addition to cytosolic targets, insulin-stimulated phospho-AKT also translocates to mitochondria in the myocardium. Mouse models of diabetes exhibit impaired mitochondrial AKT signaling but the implications of this on cardiac structure and function is unknown. We hypothesized that loss of mitochondrial AKT signaling is a critical step in cardiomyopathy and reduces cardiac oxidative phosphorylation. METHODS: To focus our investigation on the pathophysiological consequences of this mitochondrial signaling pathway, we generated transgenic mouse models of cardiac-specific, mitochondria-targeting, dominant negative AKT1 (CAMDAKT) and constitutively active AKT1 expression (CAMCAKT). Myocardial structure and function were examined using echocardiography, histology, and biochemical assays. We further investigated the underlying effects of mitochondrial AKT1 on mitochondrial structure and function, its interaction with ATP synthase, and explored in vivo metabolism beyond the heart. RESULTS: Upon induction of dominant negative mitochondrial AKT1, CAMDAKT mice developed cardiac fibrosis accompanied by left ventricular hypertrophy and dysfunction. Cardiac mitochondrial oxidative phosphorylation efficiency and ATP content were reduced, mitochondrial cristae structure was lost, and ATP synthase structure was compromised. Conversely, CAMCAKT mice were protected against development of diabetic cardiomyopathy when challenged with a high calorie diet. Activation of mitochondrial AKT1 protected cardiac function and increased fatty acid uptake in myocardium. In addition, total energy expenditure was increased in CAMCAKT mice, accompanied by reduced adiposity and reduced development of fatty liver. CONCLUSION: CAMDAKT mice modeled the effects of impaired mitochondrial signaling which occurs in the diabetic myocardium. Disruption of this pathway is a key step in the development of cardiomyopathy. Activation of mitochondrial AKT1 in CAMCAKT had a protective role against diabetic cardiomyopathy as well as improved metabolism beyond the heart.

High risk of renal outcome of metabolic syndrome independent of diabetes in patients with CKD stage 1-4: The ICKD database.

AIMS: To investigate whether metabolic syndrome (MetS) could predict renal outcome in patients with established chronic kidney disease (CKD). MATERIALS AND METHODS: We enroled 2500 patients with CKD stage 1-4 from the Integrated CKD care programme, Kaohsiung for delaying Dialysis (ICKD) prospective observational study. 66.9% and 49.2% patients had MetS and diabetes (DM), respectively. We accessed three clinical outcomes, including all-cause mortality, RRT, and 50% decline in estimated glomerular filtration rate events. RESULTS: The MetS score was positively associated with proteinuria, inflammation, and nutrition markers. In fully adjusted Cox regression, the hazard ratio (HR) (95% confidence interval) of MetS for composite renal outcome (renal replacement therapy, and 50% decline of renal function) in the DM and non-DM subgroups was 1.56 (1.15-2.12) and 1.31 (1.02-1.70), respectively, while that for all-cause mortality was 1.00 (0.71-1.40) and 1.27 (0.92-1.74). Blood pressure is the most important component of MetS for renal outcomes. In the 2 by 2 matrix, compared with the non-DM/non-MetS group, the DM/MetS group (HR: 1.62 (1.31-2.02)) and the non-DM/MetS group (HR: 1.33 (1.05-1.69)) had higher risks for composite renal outcome, whereas the DM/MetS group had higher risk for all-cause mortality (HR: 1.43 (1.09-1.88)). CONCLUSIONS: MetS could predict renal outcome in patients with CKD stage 1-4 independent of DM.

SUV3 Helicase and Mitochondrial Homeostasis.

SUV3 is a nuclear-encoded helicase that is highly conserved and localizes to the mitochondrial matrix. In yeast, loss of SUV3 function leads to the accumulation of group 1 intron transcripts, ultimately resulting in the loss of mitochondrial DNA, causing a petite phenotype. However, the mechanism leading to the loss of mitochondrial DNA remains unknown. SUV3 is essential for survival in higher eukaryotes, and its knockout in mice results in early embryonic lethality. Heterozygous mice exhibit a range of phenotypes, including premature aging and an increased cancer incidence. Furthermore, cells derived from SUV3 heterozygotes or knockdown cultural cells show a reduction in mtDNA. Transient downregulation of SUV3 leads to the formation of R-loops and the accumulation of double-stranded RNA in mitochondria. This review aims to provide an overview of the current knowledge regarding the SUV3-containing complex and discuss its potential mechanism for tumor suppression activity.

BRCA1 the Versatile Defender: Molecular to Environmental Perspectives.

The evolving history of BRCA1 research demonstrates the profound interconnectedness of a single protein within the web of crucial functions in human cells. Mutations in BRCA1, a tumor suppressor gene, have been linked to heightened breast and ovarian cancer risks. However, despite decades of extensive research, the mechanisms underlying BRCA1's contribution to tissue-specific tumor development remain elusive. Nevertheless, much of the BRCA1 protein's structure, function, and interactions has been elucidated. Individual regions of BRCA1 interact with numerous proteins to play roles in ubiquitination, transcription, cell checkpoints, and DNA damage repair. At a cellular scale, these BRCA1 functions coordinate tumor suppression, R-loop prevention, and cellular differentiation, all of which may contribute to BRCA1's role in cancer tissue specificity. As research on BRCA1 and breast cancer continues to evolve, it will become increasingly evident that modern materials such as Bisphenol A should be examined for their relationship with DNA stability, cancer incidence, and chemotherapy. Overall, this review offers a comprehensive understanding of BRCA1's many roles at a molecular, cellular, organismal, and environmental scale. We hope that the knowledge gathered here highlights both the necessity of BRCA1 research and the potential for novel strategies to prevent and treat cancer in individuals carrying BRCA1 mutations.

Loss of the oxidative stress sensor NPGPx compromises GRP78 chaperone activity and induces systemic disease.

NPGPx is a member of the glutathione peroxidase (GPx) family; however, it lacks GPx enzymatic activity due to the absence of a critical selenocysteine residue, rendering its function an enigma. Here, we show that NPGPx is a newly identified stress sensor that transmits oxidative stress signals by forming the disulfide bond between its Cys57 and Cys86 residues. This oxidized form of NPGPx binds to glucose-regulated protein (GRP)78 and forms covalent bonding intermediates between Cys86 of NPGPx and Cys41/Cys420 of GRP78. Subsequently, the formation of the disulfide bond between Cys41 and Cys420 of GRP78 enhances its chaperone activity. NPGPx-deficient cells display increased reactive oxygen species, accumulated misfolded proteins, and impaired GRP78 chaperone activity. Complete loss of NPGPx in animals causes systemic oxidative stress, increases carcinogenesis, and shortens life span. These results suggest that NPGPx is essential for releasing excessive ER stress by enhancing GRP78 chaperone activity to maintain physiological homeostasis.

Helicase SUV3, polynucleotide phosphorylase, and mitochondrial polyadenylation polymerase form a transient complex to modulate mitochondrial mRNA polyadenylated tail lengths in response to energetic changes.

Mammalian mitochondrial mRNA (mt-mRNA) transcripts are polyadenylated at the 3' end with different lengths. The SUV3·PNPase complex and mtPAP have been shown to degrade and polyadenylate mt mRNA, respectively. How these two opposite actions are coordinated to modulate mt-mRNA poly(A) lengths is of interest to pursue. Here, we demonstrated that a fraction of the SUV3·PNPase complex interacts with mitochondrial polyadenylation polymerase (mtPAP) under low mitochondrial matrix inorganic phosphate (Pi) conditions. In vitro binding experiments using purified proteins suggested that SUV3 binds to mtPAP through the N-terminal region around amino acids 100-104, distinctive from the C-terminal region around amino acids 510-514 of SUV3 for PNPase binding. mtPAP does not interact with PNPase directly, and SUV3 served as a bridge capable of simultaneously binding with mtPAP and PNPase. The complex consists of a SUV3 dimer, a mtPAP dimer, and a PNPase trimer, based on the molecular sizing experiments. Mechanistically, SUV3 provides a robust single strand RNA binding domain to enhance the polyadenylation activity of mtPAP. Furthermore, purified SUV3·PNPase·mtPAP complex is capable of lengthening or shortening the RNA poly(A) tail lengths in low or high Pi/ATP ratios, respectively. Consistently, the poly(A) tail lengths of mt-mRNA transcripts can be lengthened or shortened by altering the mitochondrial matrix Pi levels via selective inhibition of the electron transport chain or ATP synthase, respectively. Taken together, these results suggested that SUV3·PNPase·mtPAP form a transient complex to modulate mt-mRNA poly(A) tail lengths in response to cellular energy changes.

Removal of BRCA1/CtIP/ZBRK1 repressor complex on ANG1 promoter leads to accelerated mammary tumor growth contributed by prominent vasculature.

BRCA1 exerts transcriptional repression through interaction with CtIP in the C-terminal BRCT domain and ZBRK1 in the central domain. A dozen genes, including angiopoietin-1 (ANG1), a secreted angiogenic factor, are corepressed by BRCA1 and CtIP based on microarray analysis of mammary epithelial cells in 3D culture. BRCA1, CtIP, and ZBRK1 form a complex that coordinately represses ANG1 expression via a ZBRK1 recognition site in the ANG1 promoter. Impairment of this complex upregulates ANG1, which stabilizes endothelial cells that form a capillary-like network structure. Consistently, Brca1-deficient mouse mammary tumors exhibit accelerated growth, pronounced vascularization, and overexpressed ANG1. These results suggest that, besides its role in maintaining genomic stability, BRCA1 directly regulates the expression of angiogenic factors to modulate the tumor microenvironment.

Uncoupling the roles of the SUV3 helicase in maintenance of mitochondrial genome stability and RNA degradation.

Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ΔCC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ΔCC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.

Mutation of NIMA-related kinase 1 (NEK1) leads to chromosome instability.

BACKGROUND: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage. RESULTS: We show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorage-independent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates. CONCLUSION: NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

Human mitochondrial SUV3 and polynucleotide phosphorylase form a 330-kDa heteropentamer to cooperatively degrade double-stranded RNA with a 3'-to-5' directionality.

Efficient turnover of unnecessary and misfolded RNAs is critical for maintaining the integrity and function of the mitochondria. The mitochondrial RNA degradosome of budding yeast (mtEXO) has been recently studied and characterized; yet no RNA degradation machinery has been identified in the mammalian mitochondria. In this communication, we demonstrated that purified human SUV3 (suppressor of Var1 3) dimer and polynucleotide phosphorylase (PNPase) trimer form a 330-kDa heteropentamer that is capable of efficiently degrading double-stranded RNA (dsRNA) substrates in the presence of ATP, a task the individual components cannot perform separately. The configuration of this complex is similar to that of the core complex of the E. coli RNA degradosome lacking RNase E but very different from that of the yeast mtEXO. The hSUV3-hPNPase complex prefers substrates containing a 3' overhang and degrades the RNA in a 3'-to-5' directionality. Deleting a short stretch of amino acids (positions 510-514) compromises the ability of hSUV3 to form a stable complex with hPNPase to degrade dsRNA substrates but does not affect its helicase activity. Furthermore, two additional hSUV3 mutants with abolished helicase activity because of disrupted ATPase or RNA binding activities were able to bind hPNPase. However, the resulting complexes failed to degrade dsRNA, suggesting that an intact helicase activity is essential for the complex to serve as an effective RNA degradosome. Taken together, these results strongly suggest that the complex of hSUV3-hPNPase is an integral entity for efficient degradation of structured RNA and may be the long sought RNA-degrading complex in the mammalian mitochondria.

Structural requirements of chromokinesin Kif4A for its proper function in mitosis.

Human Kif4A is a member of the Kinesin-4 family of kinesins. Kif4A is thought to be a bona fide chromokinesin because it possesses a motor domain and associates with condensed chromosomes during mitosis. Genetic deletion of Kif4A promotes tumorigenic phenotypes in mouse embryonic cells. Kif4A is critical for mitotic regulation including chromosome condensation, spindle organization and cytokinesis. However, the precise chromatin-binding domain of Kif4A has not been characterized. Herein, we report the identification of two conserved motifs critical for chromatin-binding: the first leucine Zip motif (Zip1) of a leucine Zip/Basic/leucine Zip region (ZBZ) previously thought to be a nuclear localization signal (NLS), and a cysteine-rich (CR) motif within the C-terminal region of Kif4A. Furthermore, by depleting endogenous Kif4A via RNAi and concurrently expressing RNAi-resistant Kif4A versions, we observed that wild type Kif4A, but not the mutants deficient in DNA-binding (Zip1 or CR deleted) or ATPase activity (K94A point mutant), was able to rescue the RNAi-elicited abnormal mitotic profile. Taken together, our results show that both the Zip1 and CR motifs are important for Kif4A chromatin-binding and its mitotic function.

Loss of p53-regulatory protein IFI16 induces NBS1 leading to activation of p53-mediated checkpoint by phosphorylation of p53 SER37.

Our previous results that IFI16 is involved in p53 transcription activity under conditions of ionizing radiation (IR), and that the protein is frequently lost in human breast cancer cell lines and breast adenocarcinoma tissues suggesting that IFI16 plays a crucial role in controlling cell growth. Here, we show that loss of IFI16 by RNA interference in cell culture causes elevated phosphorylation of p53 Ser37 and accumulated NBS1 (nibrin) and p21WAF1, leading to growth retardation. Consistent with these observations, doxycyclin-induced NBS1 caused accumulation of p21WAF1 and increased phosphorylation of p53 Ser37, leading to cell cycle arrest in G1 phase. Wortmannin treatment was found to decrease p53 Ser37 phosphorylation in NBS-induced cells. These results suggest that loss of IFI16 activates p53 checkpoint through NBS1-DNA-PKcs pathway.

Inactivation of CtIP leads to early embryonic lethality mediated by G1 restraint and to tumorigenesis by haploid insufficiency.

CtIP interacts with a group of tumor suppressor proteins including RB (retinoblastoma protein), BRCA1, Ikaros, and CtBP, which regulate cell cycle progression through transcriptional repression as well as chromatin remodeling. However, how CtIP exerts its biological function in cell cycle progression remains elusive. To address this issue, we generated an inactivated Ctip allele in mice by inserting a neo gene into exon 5. The corresponding Ctip(-/-) embryos died at embryonic day 4.0 (E4.0), and the blastocysts failed to enter S phase but accumulated in G(1), leading to a slightly elevated cell death. Mouse NIH 3T3 cells depleted of Ctip were arrested at G(1) with the concomitant increase in hypophosphorylated Rb and Cdk inhibitors, p21. However, depletion of Ctip failed to arrest Rb(-/-) mouse embryonic fibroblasts (MEF) or human osteosarcoma Saos-2 cells at G(1), suggesting that this arrest is RB dependent. Importantly, the life span of Ctip(+/-) heterozygotes was shortened by the development of multiple types of tumors, predominantly, large lymphomas. The wild-type Ctip allele and protein remained detectable in these tumors, suggesting that haploid insufficiency of Ctip leads to tumorigenesis. Taken together, this finding uncovers a novel G(1)/S regulation in that CtIP counteracts Rb-mediated G(1) restraint. Deregulation of this function leads to a defect in early embryogenesis and contributes, in part, to tumor formation.

Aberrant DNA damage response and DNA repair pathway in high glucose conditions.

BACKGROUND: Higher cancer rates and more aggressive behavior of certain cancers have been reported in populations with diabetes mellitus. This association has been attributed in part to the excessive reactive oxygen species generated in diabetic conditions and to the resulting oxidative DNA damage. It is not known, however, whether oxidative stress is the only contributing factor to genomic instability in patients with diabetes or whether high glucose directly also affects DNA damage and repair pathways. RESULTS: Normal renal epithelial cells and renal cell carcinoma cells are more chemo- and radiation resistant when cultured in high concentrations of glucose. In high glucose conditions, the CHK1-mediated DNA damage response is not activated properly. Cells in high glucose also have slower DNA repair rates and accumulate more mutations than cells grown in normal glucose concentrations. Ultimately, these cells develop a transforming phenotype. CONCLUSIONS: In high glucose conditions, defective DNA damage responses most likely contribute to the higher mutation rate in renal epithelial cells, in addition to oxidative DNA damage. The DNA damage and repair are normal enzyme dependent mechanisms requiring euglycemic environments. Aberrant DNA damage response and repair in cells grown in high glucose conditions underscore the importance of maintaining good glycemic control in patients with diabetes mellitus and cancer.

NFBD1/Mdc1 mediates ATR-dependent DNA damage response.

Budding yeast Rad9 (scRad9) plays a central role in mediating Mec1-dependent phosphorylation by recruiting its downstream substrates. The human scRad9 orthologues 53BP1 and NFBD1 associate with ionizing radiation-induced foci (IRIF) at sites of DNA repair. RNAi-based gene silencing of 53BP1 or NFBD1 has shown impaired phosphorylation of SQ/TQ [ataxia-telangiectasia mutated/ATM and Rad3-related (ATM/ATR) substrates] at IRIF, intra-S, and G(2)-M checkpoints and has thereby revealed essential roles for 53BP1 and NFBD1 in the DNA damage signaling pathway. Whether 53BP1 and NFBD1 are required for activation of kinases and/or for recruitment of substrates at IRIF, however, is not clear. Here we show that both 53BP1 and NFBD1 are required for recruitment of ATR to DNA damage sites, as well as for ATR-dependent phosphorylation in response to DNA damage. NFBD1 is not required for ssDNA generation at DNA damage sites and is not recruited by replication protein A (RPA)-coated ssDNA. We therefore show that recruitment of NFBD1 and/or 53BP1, the factors downstream of H2AX, is independent of ssDNA generation and RPA coating, whereas both ssDNA and RPA coating play key roles in regulation of the ATR-dependent pathway. These novel findings help clarify where NFBD1 functions in DNA damage early responses.

NIMA-related protein kinase 1 is involved early in the ionizing radiation-induced DNA damage response.

Cellular functions of the NimA-related mammalian kinase Nek1 have not been demonstrated to date. Here we show that Nek1 is involved early in the DNA damage response induced by ionizing radiation (IR) and that Nek1 is important for cells to repair and recover from DNA damage. When primary or transformed cells are exposed to IR, Nek1 kinase activity is increased within 4 minutes, and Nek1 expression is up-regulated shortly thereafter and sustained for hours. At the same early time frame after IR that its kinase activity is highest, a portion of Nek1 redistributes in cells from cytoplasm to discrete nuclear foci at sites of DNA double-strand breaks. There it colocalizes with gamma-H2AX and NFBD1/MDC1, two key proteins involved very early in the response to IR-induced DNA double-strand breaks. Finally, Nek1-deficient fibroblasts are much more sensitive to the effects of IR-induced DNA damage than otherwise identical fibroblasts expressing Nek1. These results suggest that Nek1 may function as a kinase early in the DNA damage response pathway.

Deficiency of Retinoblastoma gene in mouse embryonic stem cells leads to genetic instability.

Genetic instability has been recognized as a hallmark of human cancers. Retinoblastoma (Rb) tumor suppressor protein has an essential role in modulating cell cycle progression. However, there is no direct evidence supporting its role in maintaining genetic stability. Here, we developed a sensitive method to examine the level of chromosome instability by using retrovirus carrying both positive and negative selectable markers that integrated randomly into individual chromosomes, and the frequency of loss of this selectable chromosomal marker (LOM) in normal mammalian cells was measured. Our results showed that normal mouse embryonic stem (ES) cells had a very low frequency of LOMs, which was less than 10(-8)/cell/generation. In Rb-/- mouse ES cells, the frequency was increased to approximately 10(-5)/cell/generation, whereas in Rb+/- ES cells, the frequency was approximately 10(-7)/cell/generation. LOMs was mediated mainly through chromosomal mechanisms and not through point mutations. These results, therefore, revealed that Rb, with a haploinsufficiency, plays a critical role in the maintenance of chromosome stability. The mystery of why Rb heterozygous carriers have early-onset tumor formation with high penetrance can be, at least, partially explained by this novel activity.