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.

Uncovering Minimal Pathways in Melanoma Initiation.

Cutaneous melanomas are clinically and histologically heterogeneous. Most display activating mutations in Braf or Nras and complete loss of function of one or more tumor suppressor genes. Mouse models that replicate such mutations produce fast-growing, pigmented tumors. However, mice that combine Braf activation with only heterozygous loss of Pten also produce tumors and, as we show here, in an Albino background this occurs even with Braf activation alone. Such tumors arise rarely, grow slowly, and express low levels of pigmentation genes. The timing of their appearance was consistent with a single step stochastic event, but no evidence could be found that it required de novo mutation, suggesting instead the involvement of an epigenetic transition. Single-cell transcriptomic analysis revealed such tumors to be heterogeneous, including a minor cell type we term LNM ( L ow-pigment, N eural- and extracellular M atrix-signature) that displays gene expression resembling "neural crest"-like cell subsets detected in the fast-growing tumors of more heavily-mutated mice, as well as in human biopsy and xenograft samples. We provide evidence that LNM cells pre-exist in normal skin, are expanded by Braf activation, can transition into malignant cells, and persist with malignant cells through multiple rounds of transplantation. We discuss the possibility that LNM cells not only serve as a pre-malignant state in the production of some melanomas, but also as an important intermediate in the development of drug resistance.

Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer.

CDC42 family GTPases (RHOJ, RHOQ, CDC42) are upregulated but rarely mutated in cancer and control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. Here, we use computer-aided drug design to discover a chemical entity (ARN22089) that has broad activity against a panel of cancer cell lines, inhibits S6 phosphorylation and MAPK activation, activates pro-inflammatory and apoptotic signaling, and blocks tumor growth and angiogenesis in 3D vascularized microtumor models (VMT) in vitro. Additionally, ARN22089 has a favorable pharmacokinetic profile and can inhibit the growth of BRAF mutant mouse melanomas and patient-derived xenografts in vivo. ARN22089 selectively blocks CDC42 effector interactions without affecting the binding between closely related GTPases and their downstream effectors. Taken together, we identify a class of therapeutic agents that influence tumor growth by modulating CDC42 signaling in both the tumor cell and its microenvironment.

Dynamics of nevus development implicate cell cooperation in the growth arrest of transformed melanocytes.

Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pigmented 'moles'), yet the same change is the most common driver mutation in melanoma. The reason nevi stop growing, and do not progress to melanoma, is widely attributed to a cell-autonomous process of 'oncogene-induced senescence'. Using a mouse model of Braf-driven nevus formation, analyzing both proliferative dynamics and single-cell gene expression, we found no evidence that nevus cells are senescent, either compared with other skin cells, or other melanocytes. We also found that nevus size distributions could not be fit by any simple cell-autonomous model of growth arrest, yet were easily fit by models based on collective cell behavior, for example in which arresting cells release an arrest-promoting factor. We suggest that nevus growth arrest is more likely related to the cell interactions that mediate size control in normal tissues, than to any cell-autonomous, 'oncogene-induced' program of senescence.

Melanocytes are pigment-producing cells found throughout the skin. Mutations that activate a gene called BRAF cause these cells to divide and produce melanocytic nevi, also known as "moles". These mutations are oncogenic, meaning they can cause cancer. Indeed, BRAF is the most commonly mutated gene in melanoma, a deadly skin cancer that arises from melanocytes. Yet, moles hardly ever progress to melanoma. A proposed explanation for this behavior is that, once activated, BRAF initiates a process called "oncogene-induced senescence" in each melanocyte. This process, likened to premature aging, is thought to be what causes cells in a mole to quit dividing. Although this hypothesis is widely accepted, it has proved difficult to test directly. To investigate this notion, Ruiz-Vega et al. studied mice with hundreds of moles created by the same BRAF mutation found in human moles. Analyzing the activity of genes in individual cells revealed that nevus melanocytes that have stopped growing are no more senescent than other skin cells, including non-mole melanocytes. Ruiz-Vega et al. then analyzed the sizes at which moles stopped growing, estimating the number of cells in each mole. The data were then compared with the results of a simulation and mathematical modeling. This revealed that any model based on the idea of cells independently shutting down after a number of random events could not reproduce the distribution of mole sizes that had been experimentally observed. On the other hand, models based on melanocytes acting collectively to shut down each other's growth fit the observed data much better. These findings suggest that moles do not stop growing as a direct result of the activation of BRAF, but because they sense and respond to their own overgrowth. The same kind of collective sensing is observed in normal tissues that maintain a constant size. Discovering that melanocytes do this not only sheds light on why moles stop growing, it could also help researchers devise new ways to prevent melanomas from forming.

γ-Glutamylcysteine synthetase (γ-GCS) as a target for overcoming chemo- and radio-resistance of human hepatocellular carcinoma cells.

AIMS: This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of γ­glutamylcysteine synthetase heavy chain (γ­GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo- and radio-resistance of hepatocellular carcinoma (HCC) cells. MAIN METHODS: Reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and γ­GCSh activity were measured by HPLC methods. γ­GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. KEY FINDINGS: The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding γ­GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, γ­GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Δψm and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments. SIGNIFICANCE: Our data unequivocally demonstrate that γ­GCSh may represent a prime target for overcoming anti-cancer drugs and RT resistance for HCC cells.

The RhoJ-BAD signaling network: An Achilles' heel for BRAF mutant melanomas.

Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors.

Novel roles of folic acid as redox regulator: Modulation of reactive oxygen species sinker protein expression and maintenance of mitochondrial redox homeostasis on hepatocellular carcinoma.

We provide herein several lines of evidence to substantiate that folic acid (or folate) is a micronutrient capable of functioning as a novel redox regulator on hepatocellular carcinoma. First, we uncovered that folate deficiency could profoundly downregulate two prominent anti-apoptotic effectors including survivin and glucose-regulated protein-78. Silencing of either survivin or glucose-regulated protein-78 via small interfering RNA interfering technique established that both effectors could serve as reactive oxygen species sinker proteins. Second, folate deficiency-triggered oxidative-nitrosative stress could strongly induce endoplasmic reticulum stress that in turn could provoke cellular glutathione depletion through the modulation of the following two crucial events: (1) folate deficiency could strongly inhibit Bcl-2 expression leading to severe suppression of the mitochondrial glutathione pool and (2) folate deficiency could also profoundly inhibit two key enzymes that governing cellular glutathione redox regulation including γ-glutamylcysteinyl synthetase heavy chain, a catalytic enzyme for glutathione biosynthesis, and mitochondrial isocitrate dehydrogenase 2, an enzyme responsible for providing nicotinamide adenine dinucleotide phosphate necessary for regenerating oxidized glutathione disulfide back to glutathione via mitochondrial glutathione reductase. Collectively, we add to the literature new data to strengthen the notion that folate is an essential micronutrient that confers a novel role to combat reactive oxygen species insults and thus serves as a redox regulator via upregulating reactive oxygen species sinker proteins and averting mitochondrial glutathione depletion through proper maintenance of redox homeostasis via positively regulating glutathione biosynthesis, glutathione transporting system, and mitochondrial glutathione recycling process.

ATR Mutations Promote the Growth of Melanoma Tumors by Modulating the Immune Microenvironment.

Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased T cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth.

A rare haemoglobin variant (Hb Phnom Penh) manifesting as a falsely high haemoglobin A1c value on ion-exchange chromatography.

Most haemoglobin (Hb) variants are clinically silent. However, some Hb variants may interfere with the measurement of haemoglobin A1c (HbA1c), resulting in spurious values depending on the assays used. We herein report the case of a 53-year-old Taiwanese man with type 2 diabetes mellitus, who presented with an abnormal HbA1c peak on ion-exchange chromatography. Additional investigations, including intensified self-monitored blood glucose tests, an alternative HbA1c assay, and a glycaemic indicator based on a different method, revealed that the HbA1c values were falsely elevated. Subsequent DNA analysis confirmed that the patient was heterozygous for the insertion of an isoleucine residue at codons 117/118 of the a1-globin gene, Hb Phnom Penh. Clinical laboratorians should be aware of the interfering factors in their HbA1c analysis. Cautious inspection of the chromatogram may provide a valuable clue to the presence of an Hb variant.

Increased Nek1 expression in renal cell carcinoma cells is associated with decreased sensitivity to DNA-damaging treatment.

Renal cell carcinoma (RCC) is a heterogeneous disease with resistance to systemic chemotherapy. Elevated expression of multiple drug resistance (MDR) has been suggested to be one of the mechanisms for this resistance. Here, we provide an alternative mechanism to explain RCC's resistance to chemotherapy-induced apoptosis. Never-in mitosis A-related protein kinase 1 (Nek1) plays an important role in DNA damage response and proper checkpoint activation. The association of Nek1 with the voltage-dependent anion channel (VDAC1) is a critical determinant of cell survival following DNA-damaging treatment. We report here that Nek1 is highly expressed in RCC tumor and cultured RCC cells compared to that of normal renal tubular epithelial cells (RTE). The association between Nek1 and VDAC1 is genotoxic dependent: prolonged Nek1/VDAC1 dissociation will lead to VDAC1 dephosphorylation and initiate apoptosis. Down-regulation of Nek1 expression in RCC cells enhanced their sensitivity to DNA-damaging treatment. Collectively, these results suggest that the increased Nek1 expression in RCC cells maintain persistent VDAC1 phosphorylation, closing its channel and preventing the onset of apoptosis under genotoxic insults. Based on these results, we believe that Nek1 can serve as a potential therapeutic target for drug development in the treatment of RCC.

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.

A rare hemoglobin variant (Hb Iraq-Halabja) causing spuriously low hemoglobin A1c values.

Various laboratory and patient-related factors can affect the measurement of hemoglobin A1c (HbA1c). We herein present the case of a diabetic patient with spuriously low HbA1c values on ion-exchange high-performance liquid chromatography (HPLC). Further investigations revealed that the patient was heterozygous for a rare Hb variant, namely Hb Iraq-Halabja (ß10 Ala→Val). This is the second report of this variant published in the literature. Clinicians should be aware of the limitations of HbA1c assays because inaccurate values may lead to the inappropriate management of diabetes. Unusual or discrepant HbA1c test results should prompt further investigations for potentially interfering factors, including rare Hb variants.

Oxidative stress-related enzyme polymorphisms associated with the immunological biomarkers levels in heavy drinkers in Taiwan.

BACKGROUND: Excessive alcohol intake can result in the oxidative stress in cells and the genetic variations of alcohol-metabolizing enzymes are responsible for the different degrees of toxicity of alcohol in several organs, such as the liver and immunological systems. We hypothesized that the alteration of oxidative stress due to some genetic variations of oxidative stress-related enzymes could result in changes of specific biomarkers, and heavy drinkers could be cautioned about the predictive likelihood to induce drinking-induced diseases. METHODS: A total of 108 heavy drinkers and 106 nonheavy drinkers were enrolled and the hematological, biochemical, and immunological tests were measured; the genotypes of oxidative stress-related enzymes, including manganese superoxide dismutase (MnSOD1183T>C), glutathione peroxidase 1 (GPX1Pro198Leu), catalase (CAT-262C>T), and myeloperoxidase (MPO-463G>A), were assayed by real-time polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). RESULTS: For the males, the levels of carbohydrate-deficient transferrin (CDT), malondialdehyde (MDA), CD4(+), immunoglobulin G (IgG), immunoglobulin M (IgM), and IL-6 were significantly different between the two groups. Furthermore, there were higher proportions of CD19(+) cells and lower TNF-α levels in heavy drinkers with the MnSOD C carriers, and there were higher percentages of CD19(+) cells and IL-6 levels in heavy drinkers with the combined genotypes of MnSOD C carriers and MPO A carriers. CONCLUSIONS: Our findings indicate that heavy drinkers may be cautioned predictive likelihood for them to induce drinking-induced diseases by analyzing their MnSOD genotypes and immunological biomarkers.

Novel spectrophotometric method for the quantitation of urinary xanthurenic acid and its application in identifying individuals with hyperhomocysteinemia associated with Vitamin B6 deficiency.

A novel spectrophotometric method for the quantification of urinary xanthurenic acid (XA) is described. The direct acid ferric reduction (DAFR) procedure was used to quantify XA after it was purified by a solid-phase extraction column. The linearity of proposed method extends from 2.5 to 100.0 mg/L. The method is precise, yielding day-to-day CVs for two pooled controls of 3.5% and 4.6%, respectively. Correlation studies with an established HPLC method and a fluorometric procedure showed correlation coefficients of 0.98 and 0.98, respectively. Interference from various urinary metabolites was insignificant. In a small-scale screening of elderly conducted at Penghu county in Taiwan (n = 80), we were able to identify a group of twenty individuals having hyperhomocysteinemia (>15 µ mole/L). Three of them were found to be positive for XA as analyzed by the proposed method, which correlated excellently with the results of the activation coefficient method for RBC's AST/B6 functional test. These data confirm the usefulness of the proposed method for identifying urinary XA as an indicator of vitamin B6 deficiency-associated hyperhomocysteinemic condition.

A novel small molecule RAD51 inactivator overcomes imatinib-resistance in chronic myeloid leukaemia.

RAD51 recombinase activity plays a critical role for cancer cell proliferation and survival, and often contributes to drug-resistance. Abnormally elevated RAD51 function and hyperactive homologous recombination (HR) rates have been found in a panel of cancers, including breast cancer and chronic myeloid leukaemia (CML). Directly targeting RAD51 and attenuating the deregulated RAD51 activity has therefore been proposed as an alternative and supplementary strategy for cancer treatment. Here we show that a newly identified small molecule, IBR2, disrupts RAD51 multimerization, accelerates proteasome-mediated RAD51 protein degradation, reduces ionizing radiation-induced RAD51 foci formation, impairs HR, inhibits cancer cell growth and induces apoptosis. In a murine imatinib-resistant CML model bearing the T315I Bcr-abl mutation, IBR2, but not imatinib, significantly prolonged animal survival. Moreover, IBR2 effectively inhibits the proliferation of CD34(+) progenitor cells from CML patients resistant to known BCR-ABL inhibitors. Therefore, small molecule inhibitors of RAD51 may suggest a novel class of broad-spectrum therapeutics for difficult-to-treat cancers.

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.

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR.

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G1/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1 -/- murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.

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.