Disruption of NAD(P)H:quinone oxidoreductase 1 gene in mice leads to 20S proteasomal degradation of p63 resulting in thinning of epithelium and chemical-induced skin cancer.

This corrects the article DOI: 10.1038/onc.2010.491.

Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer.

This corrects the article DOI: 10.1038/onc.2011.600.

Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer.

Previously, we have shown a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in the stabilization of p63 against 20S proteasomal degradation resulting in thinning of the epithelium and chemical-induced skin cancer (Oncogene (2011) 30, 1098-1107). Current studies have demonstrated that NQO1 control of CCAAT-enhancer binding protein (C/EBPα) against 20S proteasomal degradation also contributes to the upregulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of the NQO1 gene in mice and mouse keratinocytes led to degradation of C/EBPα and loss of p63 gene expression. p63 promoter mutagenesis, transfection and chromatin immunoprecipitation assays identified a C/EBPα-binding site between nucleotide position -185 and -174 that bound to C/EBPα and upregulated p63 gene expression. Co-immunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPα. NQO1 direct interaction with C/EBPα led to stabilization of C/EBPα against 20S proteasomal degradation. NQO1 protection of C/EBPα required binding of NADH with NQO1. Exposure of skin and keratinocytes to the chemical stress agent benzo(a)pyrene led to induction of NQO1 and stabilization of C/EBPα protein, resulting in an increase in p63 RNA and protein in wild-type but not in NQO1-/- mice. Collectively, the current data combined with previous data suggest that stress induction of NQO1 through both stabilization of C/EBPα and increase in p63 and direct stabilization of p63 controls keratinocyte differentiation, leading to protection against chemical-induced skin carcinogenesis. The studies are significant as 2-4% human individuals are homozygous and 23% are heterozygous for the NQO1P187S mutation and might be susceptible to stress-induced skin diseases.

Disruption of NAD(P)H:quinone oxidoreductase 1 gene in mice leads to 20S proteasomal degradation of p63 resulting in thinning of epithelium and chemical-induced skin cancer.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic enzyme that protects cells against chemical and radiation-induced oxidative stress and skin cancer. Disruption of NQO1 gene in mice showed thinning of skin epithelium and loss of cytokeratin 14, an early marker of skin differentiation. Immunohistochemistry and western analysis demonstrated downregulation of p63 in NQO1-/- mouse skin, as compared with wild-type (WT) mouse. Further analysis including modulation of NQO1 expression revealed a direct correlation between the levels of NQO1 and p63 in skin-derived keratinocytes and dermal fibroblasts. Modulation of proteasomal activity revealed that p63 is degraded by 20S proteasome and that this degradation is significantly rescued by NQO1. Coimmunoprecipitation studies showed that NQO1 interacts directly with p63 but not 20S to protect against this degradation. In addition, benzo[a]pyrene treatment led to induction of NQO1 and stabilization of p63 in WT but not in NQO1-/- mouse skin and keratinocytes. These data suggest that NQO1 controls stabilization of p63 and progression towards keratinocyte differentiation leading to normal skin development and presumably skin carcinogenesis.

Detection of apolipoprotein E phenotype in unconcentrated cerebrospinal fluid.

We developed a simple method to detect apolipoprotein E (Apo E) polymorphism distribution in approximately 20 microL of unconcentrated cerebrospinal fluid (CSF). A combination of isoelectric focusing in 3 M urea gel and immunoblotting was employed. Apo E phenotypes were identified in CSF samples from 45 patients with probable Alzheimer disease (AD), 15 with multiple sclerosis (MS), and 25 with other neurological diseases (OND). When the data were compared with a set of matched plasma samples, the results were identical. The method is useful for Apo E phenotyping from fresh or frozen unconcentrated CSF, when blood or plasma is not available.

Amyloid beta protein 1-40 and 1-42 levels in matched cerebrospinal fluid and plasma from patients with Alzheimer disease.

We quantitated amyloid beta proteins 1-40 (Abeta40) and 1-42 (Abeta42), and alpha1- antichymotrypsin (ACT) in matched cerebrospinal fluid (CSF) and plasma of 50 patients with probable Alzheimer disease, and analyzed the relationships with age, sex, Mini-Mental State Examination (MMSE), and apolipoprotein E phenotype. There was no relation between CSF Abeta40 and Abeta42 levels with those of plasma. CSF and plasma Abeta40 and Abeta42 levels showed no association with age, sex, and MMSE score. There was a significant correlation between CSF ACT and plasma ACT levels. The data suggest that plasma ACT crosses the blood-brain barrier. However, a lack of correlation between CSF Abeta40 and Abeta42 levels with those of plasma suggests that Abeta in CSF and plasma originates from different sources.

Increased levels of tau-like protein in patients with Down syndrome.

Tau-like protein levels from 40 Down syndrome (DS) persons (31-70 years old), 40 non-DS age-matched normal controls, 18 non-DS mentally retarded (MR) persons (26-91 years old), 25 probable Alzheimer disease (AD) patients (55-99 years old) and 24 non-demented elderly controls (54-79 years old) were measured using a sandwich enzyme linked immunosorbent assay. The levels were detected in 22 of 40 DS persons and were significantly higher in DS than any other group (P < 0.0001). There was no relationship between tau-like protein levels and age, gender or apolipoprotein E phenotypes in any of the five groups.

Interaction of scrapie agent and cells of the lymphoreticular system.

The current study focused on the role of lymphoid elements of the lymphoreticular system in scrapie pathogenesis. In the first experiment, adherent and non-adherent splenocytes from mice infected with the 139A scrapie strain were prepared. The level of infectivity on a per cell basis was significantly higher in the adherent cell population. In a second set of experiments, thymocytes, unfractionated splenocytes, T-cell enriched and T-cell depleted fractions of splenocytes were infected in vitro with ME7 scrapie strain. There was no evidence of replication of scrapie in ME7-exposed cells in any of the preparations during the first 5-14 days post-exposure. In assays done 5 days after infection, most of the infectivity was cell-associated. These data suggest that lymphoid cells are not involved in scrapie replication. The level of IgA in the serum of 139A-infected mice was markedly reduced compared to the levels in mice injected with normal mouse brain homogenate or with the ME7 scrapie strain. The reduction in IgA levels in 139A-infected mice was evident at each of the 4 time points tested. The final experiment dealt with the question of scrapie replication in the lymphoreticular organs in mouse strains with different incubation periods for 139A after intraperitoneal injection. The results in this experiment suggest that the difference in incubation periods is related to differences in time of access of infection to the central nervous system rather than to differences in the ability of agent to replicate in spleen.

Effect of treatment on oligoclonal IgG bands and intrathecal IgG synthesis in sequential cerebrospinal fluid and serum from patients with subacute sclerosing panencephalitis.

Oligoclonal IgG bands were analyzed in matching pairs of cerebrospinal fluid (CSF) and serum from 12 subacute sclerosing panencephalitis (SSPE) patients, using isoelectric focusing and immunofixation. Each patient was given isoprinosine, and four of the 12 patients were given alpha-interferon in addition. Two to 4 serial CSF and serum samples were collected from each SSPE patient during periods ranging from 1 to 16 months. In 3 SSPE patients a small number of new oligoclonal bands were seen in the follow-up CSF samples. In the other 9 SSPE patients there was no change in CSF band patterns between initial and follow-up specimens. Band patterns in serum remained unchanged between initial and follow-up samples. Although all 12 SSPE cases had higher IgG indices and increased rate of intra blood-brain barrier (BBB) IgG synthesis in comparison to patients with other neurological diseases, the values did not significantly differ between the first and follow-up specimens. We conclude that treatment of SSPE patients with isoprinosine or with isoprinosine and alpha-interferon had no significant effect on the CSF oligoclonal band profiles or IgG synthesis within the central nervous system.

Measles virus-specific immunoglobulin D antibody in cerebrospinal fluid and serum from patients with subacute sclerosing panencephalitis and multiple sclerosis.

Quantitation of measles-specific immunoglobulin D (IgD) antibody was carried out in cerebrospinal fluid (CSF) and serum samples from 18 patients with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS) and seven normal controls with high measles antibody titers in serum, using polyclonal and monoclonal antibodies specific for human IgD and enzyme-linked immunosorbent assay. Measles-specific IgD activity was significantly higher in CSF and serum from SSPE patients compared to that found in patients with MS or normal controls. The IgD antibody to measles virus was not due to high levels of measles-specific IgG since significant measles IgD activity was found after eluting IgG from SSPE serum. The increased level of measles-specific IgD found in SSPE sera is consistent with the levels observed in patients with acute and chronic viral infections.

Immunoglobulin G subclass antibodies to measles virus in patients with subacute sclerosing panencephalitis or multiple sclerosis.

Mouse monoclonal antibodies specific for human immunoglobulin G (IgG) subclasses and a sensitive immunoassay were used to evaluate the IgG subclass antibody response to measles virus antigens in cerebrospinal fluid and serum samples from 20 patients with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS), and 11 controls with high measles virus antibody titers in serum. In patients with SSPE, measles virus-specific antibodies were found mainly in the IgG1 subclass and the IgG subclass distribution remained unchanged, irrespective of the clinical stage or duration of the disease. In patients with MS and in controls, measles virus activity was also associated mainly with IgG1. However, the activity was significantly lower than that found in patients with SSPE. The results suggest that there is no primary abnormality in humoral immune response to measles virus in patients with MS. The disproportionately high levels of the measles virus-specific IgG1 subclass found in patients with SSPE may be due to persistent antigenic stimulation or reflect a defect in immunoregulatory mechanisms in response to viral infection.

Comparison of oligoclonal immunoglobulin G bands in multiple sclerosis cerebrospinal fluid by immunoblotting and immunofixation.

We previously analyzed unconcentrated cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurologic diseases by isoelectric focusing in agarose gel. We have now developed an immunoblot method for detection of oligoclonal IgG bands in unconcentrated MS CSF. The oligoclonal IgG band patterns seen after immunoblotting were compared with those of conventional immunofixation. Although immunoblotting was found to be rapid the resolution and intensity of oligoclonal IgG bands were somewhat better after immunofixation. Since immunofixation is simpler than immunoblotting, we recommend that clinical laboratories use immunofixation after isoelectric focusing to detect oligoclonal IgG bands in CSF.

Specificity of oligoclonal IgG bands against myelin proteins in chronic relapsing EAE in guinea pigs.

We showed previously by using imprint electroimmunofixation that the oligoclonal IgG in sera and CSF from chronic relapsing EAE in guinea pigs were specific to spinal cord and Mycobacterium tuberculosis. We now show that most oligoclonal IgG bands are directed predominantly against isolated myelin basic protein (MBP). Activity to the latter could be removed from sera or CSF by absorption with MBP but not with histone or lysozyme. The oligoclonal IgG reacted weakly with isolated proteolipid apoprotein, and lacked reactivity to myelin-associated glycoprotein. When the oligoclonal IgG activity to myelin proteins was removed from the sera by absorption with a preparation of delipidated myelin before imprint electroimmunofixation, a few bands in some sera still reacted with whole spinal cord homogenate. These results indicate that, in some sera, a part of the oligoclonal IgG was directed against non-myelin proteins or lipids. In contrast to chronic relapsing EAE, CSF oligoclonal IgG from patients with multiple sclerosis showed no reactivity against human brain homogenate, whole myelin, delipidated myelin, and MBP in imprint electroimmunofixation.

Detection of oligoclonal IgG bands in unconcentrated CSF by isoelectric focusing in agarose gel and silver staining.

We analyzed 102 unconcentrated cerebrospinal fluids from a variety of neurologic diseases for oligoclonal IgG bands by isoelectric focusing in agarose gel followed by staining with silver. Ten to 12 microliter of cerebrospinal fluid containing 0.4-0.8 microgram of IgG was found to be optimum. Cerebrospinal fluid from 38 of 40 patients with clinically definite multiple sclerosis, 6 of 9 with suspected multiple sclerosis and 8 of 53 patients with other neurologic diseases had oligoclonal IgG bands by IEF in agarose followed by immunofixation. The commercial system employed here is a simple sensitive and rapid method for detection of oligoclonal bands in unconcentrated cerebrospinal fluid of patients with multiple sclerosis.

Chronic relapsing EAE in guinea pigs: IgG index and oligoclonal bands in cerebrospinal fluid and sera.

IgG and albumin levels were quantitated in cerebrospinal fluid (CSF) and sera from chronic relapsing (R)-EAE animals using the enzyme linked immunosorbent assay. The animals showed increased CSF IgG compared to that of age-matched animals injected with complete Freund's adjuvant. However, the CSF IgG index suggested no significant increase in IgG synthesis within the central nervous system of R-EAE animals. Matching CSF and sera from R-EAE animals, when compared in immunofixation after isoelectric focusing, showed identical oligoclonal IgG band patterns. Although the clinical and morphologic findings in R-EAE are similar to those seen in multiple sclerosis, the site of IgG synthesis in these two diseases appears to be different.

Specificity of oligoclonal IgG bands in sera from chronic relapsing experimental allergic encephalomyelitis guinea pigs.

The specificity of oligoclonal IgG in sera from chronic relapsing EAE guinea pigs was determined by using imprint electroimmunofixation. The response of oligoclonal IgG to spinal cord and Mycobacterium tuberculosis appeared to be equal in animals sacrificed during first remission and in those sacrificed after recovery from acute EAE. In contrast, in animals sacrificed during or after the first relapse, the oligoclonal IgG seems to be directed predominantly against spinal cord. In imprint electroimmunofixation, the oligoclonal IgG specific to spinal cord did not react with guinea pig liver and kidney. In addition, activity to spinal cord could be removed from sera by absorption with spinal cord but not with kidney or liver.

Silver staining of unconcentrated cerebrospinal fluid in agarose gel (Panagel) electrophoresis.

We subjected cerebrospinal fluid (CSF) from 20 patients with multiple sclerosis and 20 patients with other neurological diseases to agarose gel ( Panagel ) electrophoresis followed by staining with silver. Ten microliters of unconcentrated CSF from multiple sclerosis patients containing 0.4 to 0.8 microgram of immunoglobulin G was found to be optimum for detection of oligoclonal IgG bands, so identified by immunofixation. The band patterns for unconcentrated CSF stained with silver were almost identical to those for the same CSF concentrated 40-fold and stained with Coomassie Brilliant Blue. Silver staining thus enables the clinical laboratory to electrophorese unconcentrated CSF on commercially prepared ( Panagel ) plates.

Absence of oligoclonal IgA in CSF and serum of multiple sclerosis patients.

CSF and serum from patients with MS and other neurologic diseases were analyzed by isoelectric focusing (IEF) in polyacrylamide gel and immunofixation with specific anti human IgA sera. The IgA band patterns seen in pH 4.5-6.0 region were rather diffuse and lacked oligoclonal feature.

Detection of oligoclonal bands in unconcentrated CSF: isoelectric focusing and silver staining.

We have developed a method to detect oligoclonal IgG bands in unconcentrated CSF from patients with MS or guinea pigs with chronic relapsing experimental allergic encephalomyelitis, by isoelectric focusing (IEF) in polyacrylamide gel and silver staining. Five to 10 microliters of CSF was sufficient. The bands were identified as IgG in immunofixation after IEF.