NDPKA is not just a metastasis suppressor - be aware of its metastasis-promoting role in neuroblastoma.

NDPK-A, encoded by nm23-H1 (also known as NME1) was the first metastasis suppressor discovered. Much of the attention has been focused on the metastasis-suppressing role of NDPK-A in human tumors, including breast carcinoma and melanoma. However, compelling evidence points to a metastasis-promoting role of NDPK-A in certain tumors such as neuroblastoma and lymphoma. To balance attention on this contrariety of NDPK-A in different cancer types, this review addresses the metastasis-promoting role of NDPK-A in neuroblastoma. Neuroblastoma is an embryonic tumor, arising from neural crest cells that fail to differentiate into the sympathetic nervous system. We summarize and discuss nm23-H1 genetics and the prognosis of neuroblastoma, structural and functional changes associated with the S120G mutation of NDPK-A, as well as the evidence supporting the role of NDPK-A as a metastasis promoter. Also discussed are the NDPK-A relevant molecular determinants of neuroblastoma metastasis, and metastasis-relevant neural crest development. Because of NDPK-A's dichotomous role in tumor metastasis as both a suppressor and a promoter, tumor genome/exome profiles are necessary to identify the molecular drivers of metastasis in the NDPK-A network for developing tumor-specific therapies.

Linking arsenite- and cadmium-generated oxidative stress to microsatellite instability in vitro and in vivo.

Mismatch repair (MMR) corrects replicative errors and minimizes DNA damage that occurs frequently in microsatellites. MMR deficiency is manifested as microsatellite instability (MSI), which contributes to hypermutability and cancer pathogenesis. Genomic instability, including MSI and chromosomal instability, appears to be responsible for the carcinogenesis of arsenic and cadmium, common contaminants in our environment. However, few studies have addressed arsenic- or cadmium-induced MSI, especially its potential link with arsenic- or cadmium-generated oxidative stress, due to the lack of quantifiable MSI assays and cost-effective animal models. Here, using a dual-fluorescent reporter, we demonstrate that sub-lethal doses of cadmium or arsenite, but not arsenate, increased the MSI frequency in human colorectal cancer cells. Arsenite- and cadmium-induced MSI occurred concomitantly with increased levels of reactive species and oxidative DNA damage, and with decreased levels of MMR proteins. However, N-acetyl-l-cysteine (NAC) suppressed arsenite- and cadmium-induced MSI and oxidative stress while restoring the levels of MMR proteins in the cells. Similarly, MSI was induced separately by arsenite and cadmium, and suppressed by NAC, in zebrafish in a fluorescinated PCR-based assay with newly-developed microsatellite markers and inter-segmental comparisons. Of five selected antioxidants examined, differential effects were exerted on the MSI induction and cytotoxicity of both arsenite and cadmium. Compared to MMR-proficient cells, MMR-deficient cells were more resistant to arsenic-mediated and cadmium-mediated cytotoxicity. Our findings demonstrate a novel linkage between arsenite-generated and cadmium-generated oxidative stress and MSI induction. Our findings also caution that antioxidants must be individually validated before being used for preventing arsenite- and cadmium-induced MSI that is associated with cancer development.

A dual-fluorescent reporter facilitates identification of thiol compounds that suppress microsatellite instability induced by oxidative stress.

The DNA mismatch-repair (MMR) system corrects replicative errors and minimizes mutations that occur at a high rate in microsatellites. Patients with chronic inflammation or inflammation-associated cancer display microsatellite instability (MSI), indicating a possible MMR inactivation. In fact, H2O2-generated oxidative stress inactivates the MMR function and increases mutation accumulation in a reporter microsatellite. However, it remains unclear whether MSI induced by oxidative stress is preventable because of the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing DsRed harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogenic human colorectal cancer cell lines. Via flow cytometry, this reporter sensitively detected H2O2-generated oxidative microsatellite mutations in a dose-dependent manner. The reporter further revealed that glutathione or N-acetylcysteine was better than aspirin and ascorbic acid for suppressing oxidative microsatellite mutations. These two thiol compounds also partially suppressed oxidative frameshift mutations in the coding microsatellites of the hMSH6 and CHK1 genes based on a fluoresceinated PCR-based assay. MSI suppression by N-acetylcysteine appears to be mediated through reduction of oxidative frameshift mutations in the coding microsatellite of hMSH6 and protection of hMSH6 and other MMR protein levels from being decreased by H2O2. Our findings suggest a linkage between oxidative damage, MMR deficiency, and MSI. The two thiol compounds are potentially valuable for preventing inflammation-associated MSI. The dual-fluorescent reporter with improved features will facilitate identification of additional compounds that modulate MSI, which is relevant to cancer initiation and progression.

Arsenic-induced health crisis in peri-urban Moyna and Ardebok villages, West Bengal, India: an exposure assessment study.

Drinking of arsenic (As)-contaminated groundwater has adverse effects on health of millions of people worldwide. This study aimed to determine the degree of severity of As exposure from drinking water in peri-urban Moyna and Ardebok villages, West Bengal, India. Arsenic concentrations in hair, nail and urine samp les of the individuals were determined. Arsenical dermatosis, keratosis and melanosis were investigated through medical evaluation. We have evaluated the association between As exposure from drinking water, and keratosis and melanosis outcomes. The results showed that 82.7 % of the sampled tube wells contain As concentrations above 10 µg/L, while 57.7 % contain As concentrations above 50 µg/L. The hair, nail and urine As concentrations were positively correlated with As concentrations in drinking water. In our study population, we observed a strong association between As concentrations ranging 51-99 µg/L and keratosis and melanosis outcomes, although the probability decreases at higher concentration ranges perhaps due to switching away from the use of As-contaminated tube wells for drinking and cooking purposes. High As concentrations in hair, nail and urine were observed to be associated with the age of the study population. The level of As concentrations in hair, nail and urine samples of the study population indicated the degree of severity of As exposure in the study region.

Identification of hnRNPH1, NF45, and C14orf166 as novel host interacting partners of the mature hepatitis C virus core protein.

The hepatitis C virus core protein (HCVc) forms the viral nucleocapsid and is involved in viral persistence and pathogenesis, possibly by interacting with host factors to modulate viral replication and cellular functions. Here, we identified 36 cellular protein candidates by one-dimensional SDS-PAGE and LC-MS/MS-based proteomics after affinity purification with HCVc174, a matured form of HCVc from HCV-1b genotype, tagged with biotin and calmodulin-binding peptide/protein A at N- and C-termini, respectively. By pull-down and confocal imaging techniques, we confirmed that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1), nuclear factor 45 (NF45), and C14orf166 are novel HCVc174-interacting host proteins, known to participate in mRNA metabolism, gene regulation, and microtubule organization, respectively. Unlike the other 2 proteins, NF45 interacted with HCVc174 in an RNA-dependent manner. These 3 proteins colocalized with ectopic HCVc-1b in both the cytoplasm and nucleus, which demonstrated their spatial interaction with naturally translocated HCVc174 after HCVc biogenesis. Such colocalization, however, shifted to the cytoplasm in cells with replicating virus of 1b or 2a genotype, indicating that active viral replication confined these interacting proteins in the cytoplasm. Collectively, our findings suggest that spatial interactions of hnRNPH1, NF45, and C14orf166 with HCVc174 likely modulate HCV or cellular functions during acute and chronic HCV infection.

Family support predicts psychiatric medication usage among Mexican American individuals with schizophrenia.

BACKGROUND: Support provided by family caregivers to persons with schizophrenia is a viable intervention focus to improve psychiatric medication usage. However, little is known about the relation between medication usage and family support as well as other key caregiving factors. METHOD: Family support and Expressed Emotion (EE) dimensions were tested as predictors of medication usage during a 9-month period following psychiatric hospital discharge in a sample of 30 individuals of Mexican descent with schizophrenia. RESULTS: Family instrumental support predicted higher medication usage (Odds Ratio = 4.8) in multivariate analyses that statistically adjusted for the impact of emotional support, family EE, and psychiatric status (e.g., positive symptoms) on medication usage. CONCLUSIONS: Findings suggest that efforts to improve medication usage among Mexican American individuals with schizophrenia should take into account social supportive factors such as instrumental or directive, hands-on assistance from family caregivers.

Nucleoside diphosphate kinase A/nm23-H1 promotes metastasis of NB69-derived human neuroblastoma.

Nucleoside diphosphate kinase A (NDPK-A), encoded by the nm23-H1 gene, acts as a metastasis suppressor in certain human tumors such as breast carcinoma. However, evidence also points to NDPK-A functioning as a metastasis promoter in other human tumors including neuroblastoma. In fact, amplification and overexpression of nm23-H1 as well as S120G mutation of NDPK-A (NDPK-A(S120G)) have been detected in 14% to 30% of patients with advanced stages of neuroblastoma. To test whether NDPK-A promotes neuroblastoma metastasis, we established stable transfectants and an orthotopic xenograft animal model from the human neuroblastoma NB69 cell line. We demonstrate that overexpressed NDPK-A or NDPK-A(S120G) increased both incidence and colonization of neuroblastoma metastasis in animal lungs without significantly affecting primary tumor development. In vitro, these metastasis-associated NDPK-A aberrations abrogated retinoic acid-induced neuronal differentiation while increasing cloning efficiency, cell survival, and colony formation of NB69 derivatives. Furthermore, NDPK-A(S120G) reduced cell adhesion and increased cell migration. Compared with its wild-type, NDPK-A(S120G) appears more effective in promoting neuroblastoma metastasis. Our results provide the first evidence that NDPK-A behaves as a metastasis promoter at least in human neuroblastoma derived from NB69 cells. The findings not only suggest a prognostic value of NDPK-A in neuroblastoma patients but also caution NDPK-A-targeted treatment for patients with different tumor types.

A fluorescent orthotopic mouse model for reliable measurement and genetic modulation of human neuroblastoma metastasis.

Neuroblastoma is the most common extra-cranial solid tumor of infancy and childhood, and majority of patients die from the metastatic disease. Orthotopic xenograft mouse models are valuable tools for improving our understanding and control of neuroblastoma metastasis, because they readily represent genetic diversity and allow spontaneous metastasis. Intra-adrenal injection is commonly used for establishing the orthotopic animal models since human neuroblastoma frequently originates in the adrenal gland. However, it is unclear whether the metastatic potential of neuroblastoma can be reliably determined in adrenally-injected mice because their gland size is so small. In this study, we developed and characterized a fluorescent orthotopic xenograft animal model of NB69-derived human neuroblastoma. By comparing animals receiving adrenal injection and adrenal overlay, with the latter mimicking injection spillover, we found that the metastatic potential of neuroblastoma can be reliably determined in animal lungs. Furthermore, the lung metastasis can be genetically modulated in these animals. The results also show that the expression of Renilla green fluorescent protein (GFP) was exceptionally stable in NB69 cells, allowing rapid and sensitive detection of lung metastases at the macroscopic level. Additional features of our model include 100% tumor take, a 1-week tumor latency, resemblance to tumor behaviors in neuroblastoma patients, and the ability to monitor the expression of a gene of interest with GFP. This animal model of human neuroblastoma will be useful for studying genes involved in the metastatic process and for evaluating anti-metastasis agents in pre-clinical trials.

Effect of H(2)O(2) on cell cycle and survival in DNA mismatch repair-deficient and -proficient cell lines.

Patients who develop tumors with Lynch syndrome, which is caused by mutational inactivation of the DNA mismatch repair (MMR) system, have a relatively favorable prognosis compared to patients who develop sporadic tumors. Paradoxically, DNA MMR-deficient cells are resistant to many chemotherapeutic agents, and are capable of bypassing the G2/M checkpoint in vitro. Colon cancers that develop in the setting of Lynch syndrome show an abundant recruitment of immune cells into tumor tissues, which might be expected to increase oxyradical formation, and make the tumor cells more vulnerable to cell death. We examined the chemosensitivity and cell cycle response to oxidative stress in several MMR-deficient (HCT116, SW48, and DLD1) and -proficient (CaCo2, SW480, and HT29) colorectal cancer cell lines. H(2)O(2) induced a G2/M cell cycle arrest in both MMR deficient and proficient cell lines, however MMR-deficient cell lines were more sensitive to H(2)O(2) toxicity, and the response was more prolonged in MMR-deficient cells. Interestingly, human MutL-homologue (hMLH1-)defective HCT116 and hMLH1-restored HCT116+ch3 cell lines responded to H(2)O(2) with the same degree of G2/M arrest. The survival response of HCT116+ch3 was nearly identical to that of hMLH1-defective HCT116+ch2, although better than the response observed in HCT116 cells. In conclusion, greater cellular sensitivity and G2/M arrest in response to oxidative stress in MMR-deficient colorectal cancer cells could be one of the reasons for the more favorable prognosis seen in patients with Lynch syndrome. However, this sensitivity appears not to be a direct result of a deficient MMR function, but is more likely attributable to spectrum of target gene mutations that occurs in MMR-deficient tumors.

Identification of frame-shift intermediate mutant cells.

Frame-shift mutations at microsatellites occur as a time-dependent function of polymerase errors followed by failure of postreplicational mismatch repair. A cell-culture system was developed that allows identification of intermediate mutant cells that carry the mutation on a single DNA strand after the initial DNA polymerase errors. A plasmid was constructed that contained 13 repeats of a poly(dC-dA).poly(dG-dT) oligonucleotide immediately after the translation initiation codon of the enhanced GFP (EGFP) gene, shifting the EGFP gene out of its proper reading frame. The plasmid was introduced into human mismatch repair-deficient (HCT116, hMLH1-mutated) and mismatch repair-proficient (HCT116+chr3, hMLH1 wild type) colorectal cancer cells. After frame-shift mutations occurred that restored the EGFP reading frame, EGFP-expressing cells were detected, and two distinct fluorescent populations, M1 (dim cells) and M2 (bright cells), were identified. M1 cell numbers were stable, whereas M2 cells accumulated over time. In HCT116, single M2 cells gave rise to fluorescent colonies that carried a 2-bp deletion at the (CA)(13) microsatellite. Twenty-eight percent of single M1 cells, however, gave rise to colonies with a mixed fluorescence pattern that carried both (CA)(13) and (CA)(12) microsatellites. It is likely that M1 cells represent intermediate mutants that carry (CA)(13).(GT)(12) heteroduplexes. Although the mutation rate in HCT116 cell clones (6.2 x 10(-4)) was 30 times higher than in HCT116+chr3 (1.9 x 10(-5)), the proportion of M1 cells in culture did not significantly differ between HCT116 (5.87 x 10(-3)) and HCT116+chr3 (4.13 x 10(-3)), indicating that the generation of intermediate mutants is not affected by mismatch-repair proficiency.

Transforming properties of a Q18-->E mutation of the microtubule regulator Op18.

We have identified a somatic mutation in Op18 in a human esophageal adenocarcinoma. The mutant form of Op18 (M-Op18) was cloned and sequenced, revealing a substitution of a G for C at nucleotide 155, which results in a Q18-->E substitution in the protein. M-Op18 cDNA was expressed in NIH/3T3 cells, which resulted in foci formation and tumor growth in immunodeficient mice. Cell cycle analysis of M-Op18-expressing cells revealed a doubling in the percentage of cells in G2/M relative to cells overexpressing wild-type Op18, a decrease in M-Op18-specific phosphorylation, and alterations in tubulin ultrastructure in M-Op18-expressing cells. These results suggest that the somatic mutation identified in Op18 has profound effects on cell homeostasis that may lead to tumorigenicity.

Oxidative stress inactivates the human DNA mismatch repair system.

In the human DNA mismatch repair (MMR) system, hMSH2 forms the hMutSalpha and hMutSbeta complexes with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the hMutLalpha heterodimer. These complexes, together with other components in the MMR system, correct single-base mismatches and small insertion/deletion loops that occur during DNA replication. Microsatellite instability (MSI) occurs when the loops in DNA microsatellites are not corrected because of a malfunctioning MMR system. Low-frequency MSI (MSI-L) is seen in some chronically inflamed tissues in the absence of genetic inactivation of the MMR system. We hypothesize that oxidative stress associated with chronic inflammation might damage protein components of the MMR system, leading to its functional inactivation. In this study, we demonstrate that noncytotoxic levels of H2O2 inactivate both single-base mismatch and loop repair activities of the MMR system in a dose-dependent fashion. On the basis of in vitro complementation assays using recombinant MMR proteins, we show that this inactivation is most likely due to oxidative damage to hMutSalpha, hMutSbeta, and hMutLalpha protein complexes. We speculate that inactivation of the MMR function in response to oxidative stress may be responsible for the MSI-L seen in nonneoplastic and cancer tissues associated with chronic inflammation.